A technique is described for forming molecular hybrids between RNA in solution and DNA in cytological preparations. Cells are immobilized under a thin layer of agar, treated with alkali to denature DNA, and then incubated with tritium-labeled RNA. Hybrids are detected by autoradiography. The technique is illustrated by the hybridization of ribosomal RNA (rRNA) to amplified rDNA in oocytes of the toad Xenopus. A low level of gene amplification was also detected in premeiotic nuclei (oogonia). Several techniques are used for annealing RNA to DNA. The DNA is often immobilized in a solid or semisolid matrix or attached to a nitrocellulose membrane. Hybrids are generally detected by scintillation counting of radioactive RNA after treatment with ribonuclease to remove unhybridized RNA. The hybridization of RNA to DNA in a cytological preparation should exhibit a high degree of spatial localization. The general principles of a cytological hybridization technique are not difficult to lay down. The chromosomes or nucleus should be fixed in as lifelike a fashion as possible; basic proteins should be removed, since they are known to interfere with the hybridization procedure; the DNA should be denatured in such a way that cytological integrity is not lost; the hybridization should be carried out with radioactive RNA of very high specific activity, since the number of hybridized molecules at a given locus will be small; and detection should be by tritium autoradiography to permit maximal cytological resolution. This communication describes a cytological hybridization technique applicable to conventional squash preparations. It is illustrated by the hybridization of rRNA to the extrachromosomal rDNA in oocytes of the toad Xenopus. A preliminary report on the technique was presented in December 1968 at the International Symposium of Nuclear Physiology and Differentiation, Belo Horizonte, Brazil. The technique combines features of the agar column and filter methods. It should be generally applicable to any material that can be examined as a squash or smear. The following procedure was used in making the preparation shown in Figure 1. The tissue was transferred to a drop of 45% acetic acid on a microscope slide and teased with jewelers' forceps. The larger bits of tissue were removed, a cover slip was added, and the cells were squashed. The slides had been previously subbed by dipping into a solution of 0.1% gelatin in 0.1% chrome alum and draining until dry. The slide was frozen on dry ice, and the cover slip was removed with a razor blade. The slides were transferred to 95% ethanol for a few moments and then dried in air. The slides were dipped in 0.5% agar held molten at 60°C in a water bath. They were removed and drained vertically at room temperature. In this way aA technique is described for forming molecular hybrids between RNA in solution and DNA in cytological preparations. Cells are immobilized under a thin layer of agar, treated with alkali to denature DNA, and then incubated with tritium-labeled RNA. Hybrids are detected by autoradiography. The technique is illustrated by the hybridization of ribosomal RNA (rRNA) to amplified rDNA in oocytes of the toad Xenopus. A low level of gene amplification was also detected in premeiotic nuclei (oogonia). Several techniques are used for annealing RNA to DNA. The DNA is often immobilized in a solid or semisolid matrix or attached to a nitrocellulose membrane. Hybrids are generally detected by scintillation counting of radioactive RNA after treatment with ribonuclease to remove unhybridized RNA. The hybridization of RNA to DNA in a cytological preparation should exhibit a high degree of spatial localization. The general principles of a cytological hybridization technique are not difficult to lay down. The chromosomes or nucleus should be fixed in as lifelike a fashion as possible; basic proteins should be removed, since they are known to interfere with the hybridization procedure; the DNA should be denatured in such a way that cytological integrity is not lost; the hybridization should be carried out with radioactive RNA of very high specific activity, since the number of hybridized molecules at a given locus will be small; and detection should be by tritium autoradiography to permit maximal cytological resolution. This communication describes a cytological hybridization technique applicable to conventional squash preparations. It is illustrated by the hybridization of rRNA to the extrachromosomal rDNA in oocytes of the toad Xenopus. A preliminary report on the technique was presented in December 1968 at the International Symposium of Nuclear Physiology and Differentiation, Belo Horizonte, Brazil. The technique combines features of the agar column and filter methods. It should be generally applicable to any material that can be examined as a squash or smear. The following procedure was used in making the preparation shown in Figure 1. The tissue was transferred to a drop of 45% acetic acid on a microscope slide and teased with jewelers' forceps. The larger bits of tissue were removed, a cover slip was added, and the cells were squashed. The slides had been previously subbed by dipping into a solution of 0.1% gelatin in 0.1% chrome alum and draining until dry. The slide was frozen on dry ice, and the cover slip was removed with a razor blade. The slides were transferred to 95% ethanol for a few moments and then dried in air. The slides were dipped in 0.5% agar held molten at 60°C in a water bath. They were removed and drained vertically at room temperature. In this way a