An impediment to the investigation of mammalian spermatogenic meiosis has been the lack of an appropriate system for experimental manipulation of meiotic prophase cells. We report here the use of a simple system for the short-term culture of pachytene spermatocytes. We have assayed parameters of cell function pertinent to meiotic prophase, namely chromosome pairing and synapsis. During the culture period of 24-48 hr, cells maintained typical pachytene morphology, chromatin condensation patterns, and chromosome pairing, as assessed by light and electron microscopy. Uridine incorporation, monitored by autoradiography, reflected the chromosomal distribution found in vivo in that the autosomal chromosomes were transcriptionally active, while the sex chromosomes were not. Thus features of chromosome pairing and sex chromatin inactivation are maintained in these cultures. We have conducted experiments to demonstrate that cultured pachytene spermatocytes can be useful for the analysis of agents, some of which may be suspected mutagens, that might affect chromosome structure and function during meiosis. Treatment of cells with actinomycin D revealed a differential effect on chromatin condensation in the autosomes versus the sex chromosomes. Camptothecin, a topoisomerase inhibitor, induced desynapsis of paired chromosomes. Okadaic acid, a phosphatase inhibitor, induced premature metaphase-I condensation of pachytene chromosomes. This last experiment suggests that these cultured cells may be useful for analysis of meiotic cell cycle controls. Taken together, these results demonstrate a culture system that can be useful for analysis of meiotic events as well as in screening for potential mutagenic agents that might affect meiotic chromosome structure and function.