Monoclonal antibodies MRC OX-45 and OX-46 detect identical or juxtaposed antigenic determinants on a novel rat membrane molecule that plays a possible role in macrophage suppression of antigen-induced T-cell responses. These antibodies react with most mature hematopoietic cells and their bone-marrow precursors, vascular endothelium and some connective tissue. The OX-45 antigens were purified from brain (mainly endothelium) and spleen by immunoaffinity chromatography, and were found to be glycoproteins with apparent Mr 43,000 and 45,000, respectively, as determined by SDS-PAGE analysis. The amino acid compositions of the two preparations were very similar but with no distinguishing features. The broad pattern of distribution was not the result of fortuitous cross-reaction of the MAbs as a single N-terminal sequence was obtained from mixed spleen populations of cells. Carbohydrate compositions of the brain and spleen molecules differed both in absolute amount (22 and 41% by weight, respectively) and in the ratios of various saccharides reflecting overall differences in the patterns of glycosylation between the two tissues. MRC OX-45 IgG showed an heterogeneity in the Mr of its H chain due to the attachment, in some molecules, of carbohydrate structures to the Fd fragment.