A quantitative analysis of the proliferative response induced in murine thymocytes by concanavalin A (Con A) is described. Exogenous 3H-thymidine labels 35 to 40% of the newly incorporated TMP residues under optimal conditions. The density label 5-bromo-2-deoxuridine (BrUdR) does not affect DNA metabolism in this system. With this nucleoside, it is shown that newly synthesized DNA is the result of semi-conservative replication, not repair. Double labeling of DNA provides a monitor for cells traversing the cell cycle (S phase to subsequent S phase). The average cycle time is 12.5 hr, and the shortest cell cycle time is 10 hr. The growing fraction of active cells is about two-thirds. The data show that different subpopulations of thymocytes begin proliferating after various times in culture. Once effectively stimulated by Con A, some of the cells can traverse the cell cycle at least twice more after the mitogen is removed.