Testicular cells were prepared from neonatal (48 h after birth) mice by enzymatic dissociation and were cultured in serum-supplemented medium to investigate cell proliferation in vitro. The cultured cells were composed mostly of germ cells, identified by immunocytochemistry using a germ cell-specific antiserum, and supporting (immature Sertoli) cells. After 36 h in culture, the cells were pulse-labeled with 3H-thymidine and fixed at 2-h intervals for 36 h after labeling. Numbers of labeled and unlabeled metaphases of germ cells and supporting cells were counted, and percent labeled metaphases for both cell types were determined for cell-cycle analysis. The results indicate that germ cells, as well as supporting cells, incorporate 3H-thymidine and progress through the cell cycle in vitro. From the curve of the percent labeled metaphases for the supporting cells, the total cell cycle and intervals of DNA synthesis were estimated to be 27.2 h and 13.2 h, respectively.