We have previously described a unique model of long-term, multilineage, human hematopoietic chimerism in sheep created by the in utero transplantation of human hematopoietic stem cells (HSC) into pre-immune fetal lambs. In this study, we examined the effect of chronic administration of recombinant human mast cell growth factor (rhMGF) on 1) human cell engraftment in pre-immune sheep and 2) human cell expression in human-sheep chimeras at 2-years posttransplant. rhMGF (25 micrograms/kg) or saline was administered in utero via chronic intraperitoneal (IP) catheters to three separate sets of twin fetuses on alternate days for 10 doses following transplantation of human HSC. Flow-cytometric and karyotype analyses of peripheral blood from two sets of twins at 45-days posttransplant and of peripheral blood from the remaining set of twins at birth revealed a significant increase in percentages of donor (human) progenitors and cells in rhMGF-treated lambs. rhMGF (60 micrograms/kg/day) was also administered by IP injection to two, 2 year-old, human-sheep chimeras for 18 consecutive days. Flow-cytometric analysis of peripheral blood and bone marrow revealed a six- to seven-fold increase in human cell expression. The effect on early human progenitors (i.e., colony-forming unit-mix [CFU-Mix], CFU granulocyte/macrophage [CFU-GM], and burst-forming unit-erythroid [BFU-E]) was determined by karyotype analysis of individual colonies grown under conditions favoring human cell growth. A three- to five-fold increase in human CFU-Mix and BFU-E occurred with a minimal increase in CFU-GM. This in vivo study supports in vitro data suggesting that MGF is a powerful regulator of human hematopoiesis and preferentially stimulates early hematopoietic progenitors. It also supports the potential value of the human-sheep model for the in vivo study of normal and abnormal human hematopoiesis.