Flt-1 is a high affinity binding receptor for the vascular endothelial cell growth factor (VEGF) and is primarily expressed in endothelial cells. In this study we have investigated the temporal and spatial regulation of its expression by establishing mouse lines containing the lacZ gene targeted into the flt-1 locus through homologous recombination in embryonic stem (ES) cells. In the yolk sac as well as in the embryo proper, lacZ expression faithfully reflected the endogenous expression pattern of the flt-1 gene. LacZ staining of heterozygous embryos led to the following observations: (1) the onset of flt-1 expression is detected at the early primitive streak stage in the extraembryonic mesoderm, and is strongly up-regulated thereafter, reaching a maximum by early to midsomite stages and declining subsequently; (2) while flt-1 is widely expressed within the developing vascular endothelium, its expression level is differentially regulated both spatially and temporally. The pattern of flt-1 expression suggests that it may play an important role in the initiation of endothelium development; and (3) flt-1 is expressed in essentially all the cells in early blood islands, but later its expression is gradually restricted to the endothelial lineage. Our results indicate that flt-1 is a marker for hemangioblasts, the presumed progenitor for both hematopoietic and angioblastic lineage. The flt-1 expression pattern also suggests that it may play important roles in both vasculogenesis and angiogenesis.