A phagemid system was developed for the monovalent display of combinatorial antibody Fab libraries on the surface of filamentous phage M13. Fab fragments were fused to the carboxyl-terminal domain of the gene III protein. Phage displaying Fab fragments on their surface, or Phabs, were enriched by 10³- to 10⁵-fold on antigen-coated surfaces over nonspecific phage. This method may replace current antibody cloning techniques. The combinatorial approach provides a means of capturing the vast diversity of the immunological repertoire. The approach relies on the ability to clone antibody heavy- and light-chain fragments independently and randomly recombine them in a system that allows the specificity of binding to be probed. Previously, a λ phage system was used for probing plaque-lifts for the identification of desired clones. This system is limited by the size of the combinatorial library that may be examined (≈10⁶ members). An interesting approach to accessing larger libraries involves their expression on the surface of filamentous phage. The display of libraries of small peptides on the surface of filamentous phage has proven to be a powerful approach for selecting ligands of defined specificity. Phage display libraries are rapidly being extended to whole proteins. Two monomeric proteins, a single-chain antibody and human growth hormone, have been successfully expressed as fusions with the gene III (gIII) product coat protein III (cpIII). We have recently reported the expression and assembly of considerably larger and more complex (≈50-kDa heterodimers) proteins, antibody Fab fragments, as fusions with the gene VIII (gVIII) product coat protein VIII (cpVIII). The ability to rapidly sort large combinatorial Fab libraries is particularly important for the development of catalytic antibodies. Here we report a strategy based on gIII fusions, complementing our gVIII approach, for the construction, selection, and production of high-affinity antigen-specific Fabs from combinatorial antibody libraries. The combinatorial antibody technology reported here may replace current hybridoma methods for the isolation of monoclonal antibodies. The pComb 3 vector was designed for rapid conversion from a selection vector to an expression vector. This facilitated the preparation of soluble Fab at a level estimated to be several milligrams per liter depending on the clone. Three positive clones selected from the antitetanus toxoid library were examined by competitive ELISA, and the expected antigen specificity was confirmed. The apparent binding affinities of the isolated clones are on the order of those isolated using the λ phage system. We have demonstrated that monovalent Fab phage display libraries can be constructed, and specific binders can be rapidly selected. The reduced valency of displayed molecules allows the experimenter not only to isolate clones of defined specificity, but to select for higher affinity binders mimicking B-cell selection in a laboratory setting. This hasA phagemid system was developed for the monovalent display of combinatorial antibody Fab libraries on the surface of filamentous phage M13. Fab fragments were fused to the carboxyl-terminal domain of the gene III protein. Phage displaying Fab fragments on their surface, or Phabs, were enriched by 10³- to 10⁵-fold on antigen-coated surfaces over nonspecific phage. This method may replace current antibody cloning techniques. The combinatorial approach provides a means of capturing the vast diversity of the immunological repertoire. The approach relies on the ability to clone antibody heavy- and light-chain fragments independently and randomly recombine them in a system that allows the specificity of binding to be probed. Previously, a λ phage system was used for probing plaque-lifts for the identification of desired clones. This system is limited by the size of the combinatorial library that may be examined (≈10⁶ members). An interesting approach to accessing larger libraries involves their expression on the surface of filamentous phage. The display of libraries of small peptides on the surface of filamentous phage has proven to be a powerful approach for selecting ligands of defined specificity. Phage display libraries are rapidly being extended to whole proteins. Two monomeric proteins, a single-chain antibody and human growth hormone, have been successfully expressed as fusions with the gene III (gIII) product coat protein III (cpIII). We have recently reported the expression and assembly of considerably larger and more complex (≈50-kDa heterodimers) proteins, antibody Fab fragments, as fusions with the gene VIII (gVIII) product coat protein VIII (cpVIII). The ability to rapidly sort large combinatorial Fab libraries is particularly important for the development of catalytic antibodies. Here we report a strategy based on gIII fusions, complementing our gVIII approach, for the construction, selection, and production of high-affinity antigen-specific Fabs from combinatorial antibody libraries. The combinatorial antibody technology reported here may replace current hybridoma methods for the isolation of monoclonal antibodies. The pComb 3 vector was designed for rapid conversion from a selection vector to an expression vector. This facilitated the preparation of soluble Fab at a level estimated to be several milligrams per liter depending on the clone. Three positive clones selected from the antitetanus toxoid library were examined by competitive ELISA, and the expected antigen specificity was confirmed. The apparent binding affinities of the isolated clones are on the order of those isolated using the λ phage system. We have demonstrated that monovalent Fab phage display libraries can be constructed, and specific binders can be rapidly selected. The reduced valency of displayed molecules allows the experimenter not only to isolate clones of defined specificity, but to select for higher affinity binders mimicking B-cell selection in a laboratory setting. This has