Significant morbidity and mortality are associated with the conditioning therapy needed for postnatal bone marrow transplantation (BMT) for inherited diseases. This could be eliminated with hematopoietic stem cell (HSC) transplantation in utero, when the immunoincompetence of the fetus permits engraftment without the need for immunosuppressive therapy. We have established an in utero (day 11 to day 13) model of HSC transplantation in nondefective, allogeneic major histocompatibility complex (MHC)-mismatched mice. Donor cells wre from pooled fetal livers of C57BL/6 (H-2b, GPI-1b) mice. Engraftment was tested by quantitative polymerase chain reaction (PCR) for the Y chromosome in female recipients (with 0.00001% sensitivity). Eight percent (3 of 36) of allogeneic mismatched (Balb-c, H-2d) recipients and 25% (3 of 12) of congenic (C57B1/6, GPI-1a) recipients showed durable engraftment (male donor cells detected beyond 20 weeks of age) based on analysis of peripheral blood leukocytes (P > .08). When spleen and liver were analyzed, 51% (17 of 33) of allogeneic recipients and 50% (6 of 12) of congenic recipients showed durable engraftment (P > .3). The percent donor cells that durably engrafted varied from as low as 0.0001% in spleen and liver to as high as 0.6% in peripheral blood. Postnatal boosting with a single dose of allogeneic MHC-mismatched donor cells in a tolerant, engrafted mouse resulted in a significant increase in donor cells in the peripheral blood from 0.2% pre-boost to 5% 6 months after the boost. There was no evidence of engraftment in nontolerant mice after the postnatal boost with a similar dose of donor cells. Twenty-two allogeneic recipients were evaluated for donor skin graft acceptance at 6 to 12 months of age. Three mice with engraftment in blood and/or tissue permanently accepted donor skin grafts, one of them with donor cells detectable only in the liver. Six additional mice that showed prolonged skin graft acceptance had no evidence of durable engraftment in the blood but were engrafted in the liver and/or spleen. The degree of engraftment in tolerant mice was low (< or = 0.1% donor cells). We conclude that at an early gestational age in nondefective mice (1) high rates of durable engraftment are achievable, although the degree of engraftment is usually low (less than 1%); (2) the percent of donor cells in the peripheral blood may be increased by a postnatal boost of donor cells in tolerant animals without conditioning therapy; (3) MHC appears to have little influence on engraftment efficiency at an early gestational age; (4) a very small number of circulating donor cells in the blood or the tissues is sufficient for the induction and maintenance of tolerance, and (5) the presence of donor cells in the circulating blood is not necessary for prolonged skin graft acceptance or maintenance of permanent skin graft acceptance.