Fibroblast surface (SE) antigen is present in fibrillar surface structures of cultured normal fibroblasts, shed to the extracellular medium, and is also found in circulation (serum and plasma). Malignant fibroblasts (transformed by viruses) do not express SF antigen on the cell surface. In this study the in vivo differentiation and distribution of SF antigen has been investigated in the developing chick embryo using cryostat sections and immunofluorescence. The major findings were: (a) SF antigen was detectable in the loose connective tissue of very early (2-to 3-day old) embryos. (b) Condensation of SF antigen was seen in various boundary membranes such as the glomerular and tubular basement membranes of the kidney, the boundary membranes of the notochord, yolk sac, and vitelline membranes and liver sinusoids. (c) SF antigen was found to be cell-type specific. It was seen as a fibrillar network in the loose connective tissue of different organs but not in the parenchymal cells. It was not found in muscle cells at any stage of development. (d) The antigen was present in the undifferentiated mesenchymal cells of the kidney; but not found after their development into epithelial cells of the secretory tubules. (e) Both in vivo and in fibroblast cultures SF antigen was distributed as a fibrillar network. These data indicate that SF antigen is a "differentiation antigen" restricted to certain cells of mesenchymal origin and character, and that is accumulates in the connective tissue during embryogenesis.