We have demonstrated by indirect immunofluorescence the cellular localization of a monoclonal antibody (mAb 224-58), produced after immunization of a mouse with human central nervous system (CNS) myelin. Serologically, mAb 224-58 was found to be specific for 3'-sulfomonogalactosylglycolipids, namely 3'-sulfogalactosylceramide (SGC) and 3'-sulfogalactosyl 1-O-alkyl ether 2-O-acylglycerol (seminolipid). This mAb did not bind to SGC-containing tissues such as kidney, liver, spleen, or brain, nor to muscle. However mAb 224-58 did stain positively mouse, rat, and human peripheral nerve sections. In these latter sections, mAb 224-58 was bound to Schwann cell bodies and processes. The specificity of mAb 224-58 for Schwann cells was ascertained on teased rat sciatic nerves and rat Schwann cell cultures. Cells positive for mAb 224-58 were also positive for laminin, and negative for Thy 1-1 antigens both in teased fibers and Schwann cell cultures. In addition, in teased nerve preparations, mAb 224-58-positive cells were also galactosylceramide (GalC)- and SGC-positive. Isolated Schwann cells also expressed 224-58 antigen, even after prolonged time in culture. On testis sections, which contain both SGC and seminolipid, the SGC-positive cells, i.e., the spermatogonia, were always 224-58-negative. But the other germinal cells were 224-58-positive. This suggests that although 224-58 does not discriminate between SGC and seminolipid in serological tests, these lipids in their naturally occurring membrane acquire a spatial configuration that renders them distinguishable to their respective antibody.