Cells that generate splenic colonies within 8 days (day-8 colony- forming units-spleen [CFU-s]) are generally thought to differentiate only into erythroid/myeloid cells. The T and B lymphocyte differentiation potentials of day-8 CFU-s were evaluated and compared with those of day-12 and 5-fluorouracil (5-FU) CFU-s. This was achieved by analyzing, after intravenous and intrathymic injection, the lymphocyte progeny of cells contained within individual splenic colonies collected at day 8 and day 12 post-bone marrow cell transfer into irradiated congenic recipients. A large majority of day-8 spleen colonies generated T cells when transferred intrathymically. After intravenous (IV) injection of day-8 colonies, donor-type thymocytes emerged in 33% of the animals reconstituted with only 1 day-8 colony, but in 83% of those inoculated with a pool of 5 colonies. All post-5-FU and 75% of day-12 colonies gave rise to thymocytes after IV injection. B cells were generated by a high proportion of day-8 colonies, and by all day-12 and post 5-FU colonies. These results demonstrate that progenitors of T and B lymphocytes are generated within spleen colonies produced by at least some day-8 CFU-s and virtually all day-12 CFU-s. Whether these progenitors are CFU-s themselves or committed precursors remains an open question.