A simple technique is described for preparing collagen substrata containing 0.1% collagen by weight, in the form of native bundles with a 640 Å period, similar to soft-tissue matrices. These substrata are hydrated collagen lattices (HCLs), where the watery milieu is held within a fibrous collagen net mainly by capillary forces. HCLs have been characterized in terms of collagen precipitation and aggregation, ultrastructure, and stability under various conditions. The use of HCLs as two- and three-dimensional substrata in cell behavioral studies is illustrated with preliminary observations on the form, motility, adhesion, and growth of human diploid cells and two lines of malignant cells. The preparation of HCLs involves precipitating collagen and aggregating it into dispersed native bundles within a fluid having the same composition as the tissue culture medium. The collagen solutions are stable at low ionic strength and low pH. The preparation of HCLs is simple, cheap, and requires no special skills. HCLs are best prepared within a day of use and kept in airtight bags at room temperature. They are unstable at low temperatures, losing water. Trypsin has no effect on HCLs; traces of bacterial collagenase lead to rapid dissolution. HCLs have been used in three ways: as a two-dimensional substratum, to overlay cells already attached to a substratum, and as a three-dimensional substratum incorporating cells. Mix can be poured or spread over the surface of a dish to provide substrata of a desired area and thickness. The draining technique can be used to align the collagen bundles if desired. Cells respond to much lower concentrations of collagen in their environment. Dilute collagen overlays are simply made by mixing a small quantity of collagen into the existing fluid medium. Preliminary observations show that cells plated onto HCLs behave superficially much as they would on plastic. However, the form of the cells in sparse culture is quite different. Seeded sparsely onto HCLs, fibroblasts characteristically adopt the bipolar spindle form. The cell is greatly extended with a clear differentiation between pseudopodial and nonpseudopodial surface. The predominant form remains the bipolar spindle. Normal fibroblasts adhere strongly to HCLs. Confluent membranes of fibroblasts investing drop lattices cannot be removed by shear. A standard 0.25% trypsin solution used for subculturing causes cells to detach from plastic in less than 15 min at 37°C. This treatment has no effect on cells attached to an HCL. After 1 or 2 hr there is a general shortening of the cells, some of which are no longer firmly attached. After 12 hr all cells are detached and moribund. Cells overlaid by an HCL tend to release their attachments to the plastic and move a little way into theA simple technique is described for preparing collagen substrata containing 0.1% collagen by weight, in the form of native bundles with a 640 Å period, similar to soft-tissue matrices. These substrata are hydrated collagen lattices (HCLs), where the watery milieu is held within a fibrous collagen net mainly by capillary forces. HCLs have been characterized in terms of collagen precipitation and aggregation, ultrastructure, and stability under various conditions. The use of HCLs as two- and three-dimensional substrata in cell behavioral studies is illustrated with preliminary observations on the form, motility, adhesion, and growth of human diploid cells and two lines of malignant cells. The preparation of HCLs involves precipitating collagen and aggregating it into dispersed native bundles within a fluid having the same composition as the tissue culture medium. The collagen solutions are stable at low ionic strength and low pH. The preparation of HCLs is simple, cheap, and requires no special skills. HCLs are best prepared within a day of use and kept in airtight bags at room temperature. They are unstable at low temperatures, losing water. Trypsin has no effect on HCLs; traces of bacterial collagenase lead to rapid dissolution. HCLs have been used in three ways: as a two-dimensional substratum, to overlay cells already attached to a substratum, and as a three-dimensional substratum incorporating cells. Mix can be poured or spread over the surface of a dish to provide substrata of a desired area and thickness. The draining technique can be used to align the collagen bundles if desired. Cells respond to much lower concentrations of collagen in their environment. Dilute collagen overlays are simply made by mixing a small quantity of collagen into the existing fluid medium. Preliminary observations show that cells plated onto HCLs behave superficially much as they would on plastic. However, the form of the cells in sparse culture is quite different. Seeded sparsely onto HCLs, fibroblasts characteristically adopt the bipolar spindle form. The cell is greatly extended with a clear differentiation between pseudopodial and nonpseudopodial surface. The predominant form remains the bipolar spindle. Normal fibroblasts adhere strongly to HCLs. Confluent membranes of fibroblasts investing drop lattices cannot be removed by shear. A standard 0.25% trypsin solution used for subculturing causes cells to detach from plastic in less than 15 min at 37°C. This treatment has no effect on cells attached to an HCL. After 1 or 2 hr there is a general shortening of the cells, some of which are no longer firmly attached. After 12 hr all cells are detached and moribund. Cells overlaid by an HCL tend to release their attachments to the plastic and move a little way into the