The ontogeny of development of precursors of cytolytic T lymphocytes (CTL-P) in the murine thymus has been investigated. Fetal or newborn thymocytes harvested from C57BL/6 mice were cultured with irradiated allogeneic (DBA/2) spleen cells in limiting dilution mixed leukocyte microcultures and assayed for cytotoxicity against 51Cr-labeled P-815 (DBA/2) tumor target cells. By applying Poisson statistics to these data, minimal estimates of thymic CTL-P frequencies were determined. Whereas CTL-P were barely detectable in fetal or newborn thymuses (3-19 CTL-P/10(6) cells), their frequency increased dramatically (to 44-127 10(6) cells) within 24 h of birth. In comparison, control adult (day 70) thymuses contained 300-400 CTL-P/10(6) cells. Mixing experiments indicated that the low CTL-P frequencies observed in newborn thymuses could not be attributed to a suppressive phenomenon. These data provide strong indirect evidence for functional maturation of CTL-P within the neonatal thymus.