Hemopoiesis, initiated in the early embryo yolk sac (YS) (7.5-8 days postcoitum (pc) in mouse), takes place thereafter in sites successively seeded by extrinsic hemopoietic stem cells (HSC). Since the existence of intraembryonic HSC has been proven experimentally in some vertebrates, it is also likely that not all HSC originate in the YS in mammals, as previously thought. Candidate intraembryonic sites that may be active in producing HSC before liver colonization are the para-aortic splanchnopleura (P-Sp) and the aorta-gonads-mesonephros region (AGM). Here we explore these sites directly for the presence of cells with hemopoietic-specific surface molecules and gene activities. The Ags c-kit, AA4.1, Mac-1, and Sca-1 begin to be expressed on some P-Sp and AGM cells, making it possible to distinguish subpopulations that evolve according to reproducible developmental patterns. On the basis of RAG-1 gene transcription, the first lymphoid precursors in the mouse embryo appear to be present 9.5 to 10 days pc in P-Sp/AGM and YS. Starting B-cell lymphopoiesis (9-12 days pc) is characterized by nonexpression of the surrogate light chain lambda 5-encoding gene and biased usage of IgH DJ4 rearrangements. In the 12.5- to 13.5-day-pc fetal liver (FL), a switch occurs, characterized by the random use of all IgH DJ and the detection of lambda 5 gene transcripts.