The human cell surface molecule CD34 is selectively expressed on uncommitted and committed hematopoietic progenitor cells and on vascular endothelial cells. It has been suggested that CD34 regulates early events in blood cell migration and differentiation, possibly as a cell adhesion molecule. To characterize the patterns of expression of CD34 in the mouse embryo and in the adult, as well as to dissect the function of different portions of the extracellular domain of this molecule, we have generated the first monoclonal antibodies (mAb) specific for mouse CD34. The epitope(s) recognized by these mAb are not carbohydrate moieties, and are comprised either within the immunoglobulin-like domain or within a portion of the mucin domain, containing approximately half of the predicted O- and N-linked carbohydrate attachment sites. The specificity of the antibodies was established by ELISA and Western blotting. Western analysis revealed that these mAb recognize a protein of approximately 110 kDa in PA6 stromal cell lysates, which can be specifically blocked by the recombinant CD34 protein. To establish the reactivity of these mAb on different cell lineages, a panel of cell lines was stained. This analysis showed strong reactivities with 3T3 fibroblasts, stromal cell lines from fetal liver and with the endothelial cell line D10. Bone marrow hematopoietic progenitors were also stained by these mAb. Immunostaining of frozen sections from embryonic and adult tissues revealed a strong reactivity against vascular endothelial cells at different stages of development, including sinusoidal cells in the fetal liver, yolk sac, and in the fetal bone marrow, endothelial cells from adult lung and kidney, and neural cells, including those of the neural tube of midgestation embryos and neuronal bodies in adult brain.