Thymic and splenic lymphocytes of 6- to 8-week-old chickens responded well to the mitogens PHA and ConA in semimicro cultures using serum-free RPMI 1640 medium. Embryonic thymic lymphocytes, derived from 20-day-old embryos or the thymic anlagen of 10-day-old embryos after 10 days in organ culture, showed relatively low, but significant, in vitro responses to both PHA and ConA. The optimal concentration of PHA was generally higher for thymic cells than for splenic cells. Furthermore, while the embryonic thymic lymphocytes responded best at high ConA concentrations, thymic and splenic cells from the young chickens gave peak responses at lower ConA concentrations.