Hybridoma cell lines were generated producing monoclonal antibodies to chick gp 115, a 115,000-dalton glycoprotein widely distributed in the connective tissue. The specificity of the antibodies was determined by indirect radioimmunobinding: the extent of binding was a function of i) antigen and ii) antibody concentration; iii) inhibition of binding of radiolabelled antibody by unlabelled antibody and iv) among many known extracellular collagenous or noncollagenous glycoproteins tested only gp 115 gave a strong positive binding reaction. The antibodies were used for indirect immunofluorescence and a strong staining reaction was detected in all blood vessels, around smooth muscle cells in several organs, and in the connective matrix of other tissues such as the liver, and the lung. Based on the competition of binding of [125I]-labeled purified antibody by unlabeled antibodies, two separate epitopes were identified on gp 115. Further analysis of the localization of the epitope was obtained by CNBr cleavage and partial digestion of gp 115 with Staphylococcus aureus V8 protease and alpha-chymotrypsin digestion. Following CNBr cleavage a major fragment of Mr = 35,000 was recognized by 4 monoclonal antibodies, and fragments of comparable Mr were detected following V8 protease and alpha-chymotrypsin digestion.