The existence of B-cell and T-cell lymphocytes in chelonians is inferred by their ability to make antibodies and reject grafts. In Vitro evidence based on the ability of subpopulations to respond to a R-cell mitogen (LPS) and T-cell mitogens (Con A and PHA) is lacking. Thymus and spleen were collected from 3-4 year old cultured turtles, pressed through a wire grid and suspended in Eagles high amino acid media. The buffy coat from peripheral blood and the suspensions of thymocytes and splenocytes were layered onto Percol for centrifugation to isolate mononuclear cells. The washed cells were adjusted to a final concentration of 1 x 10⁶ cells per flatbottom well. Various concentrations of LPS, Con A and PHA were added to the wells. After 72 hours incubation in 5% C02 at 30'C, the cells were pulsed with tritiated thymidine. The cells were harvested through day 9, and the counts per minute determined by a beta counter. The cells from each compartment were able to respond to the mitogens. The thymocytes peaked at day 7 for Con A but exhibited decreased responsiveness to PHA by day 5. The splenocytes peaked at day 5 for Con A, PHA and LPS. The peripheral blood peaked at days 7-9 to Con A and LPS, but exhibited decreased responsiveness to PHA after day 5. The responsiveness of these immunocytes to various mitogens indicates that different subpopulations of lymphocytes are present in Chelonia mydas.