There is evidence that glycans carrying terminal galactose residues are differently expressed in the sarcoplasm of different muscle fiber types. In this study monoclonal antibodies directed against P blood group antigens Pk: Galalpha1-4Galbeta1-4Glcbeta- and P1: Galalpha1-4Galbeta1-4GlcNAcbeta- were used to detect terminal alpha-galactosylated glycoconjugates on muscle proteins. Electrotransfer of proteins, extracted from human masseter and biceps muscles, to nitrocellulose after polyacrylamide gel electrophoresis (PAGE) and incubation with anti-Pk (CD77) consistently showed two bands with apparent molecular weights of 66 kDa and 64 kDa. In fresh frozen muscle sections from some humans there was endothelial reaction with anti-CD77 in capillaries, venules and veins but not in arterioles and arteries. In muscle samples from other humans there was no staining of endothelial cells. Formalin-fixed human muscle displayed a CD77 reaction with highest accumulation of reaction product at the periphery of the fibers. This may be explained by the presence of Pk glycoconjugates on intermediate filaments in muscle fibers. In preparations of cat masseter muscle proteins the antibodies against P1Pk antigens reacted with a 170 kDa and a 55 kDa band while in preparations of cat biceps brachii only a 55 kDa band was reactive. The specificities of the antibodies were investigated by fluorescence-activated cell sorter (FACS), alpha- and beta-galactosidase digestion and inhibitory sugars. This study indicates that glycans carrying Galalpha1-4Galbeta1- epitopes are expressed on myofibrillar associated proteins.