The mechanism of spleen colony formation in nonirradiated mice was investigated. When the spleen cells of C57BL mice immunized against CBA- T6T6 mice were injected into the nonirradiated BT6F1 (C57BL X CBA-T6T6) hybrid mice, the number of hematopoietic stem cells (CFU-S of C57BL mouse origin that settled in the spleen of the BT6F1 mice continued to decrease in the first 9 days and then started to increase, with a doubling time of about 36 hr. Colonies were detected on the surface of the spleen 16–22 days after the cell injection. The slower appearance of spleen colonies in nonirradiated mice (compared with 6–10 days in the irridiated animals) appears to be due to retarded start of differentiation and to the prolonged doubling time of CFU-S in non- irradiated mice.