Sarcospan is a 25 kDa transmembrane component of dystrophin-associated glycoprotein. We generated a rabbit polyclonal antibody against synthetic peptide of the N-terminal domain of human sarcospan. Using this antibody we investigated the localization of sarcospan and its spacial relation to the components of sarcoglycan subcomplex in normal human skeletal myofibers by immunofluorescent microscopy and immunogold electron microscopy. In immunofluorescence the reaction was observed continuously at the myofiber surface. Ultrastructurally the gold signals of rabbit anti sarcospan antibody were present along the muscle plasma membrane, mainly at its inside surface. The triple immunogold labeled muscle samples showed that the signals of rabbit or sheep polyclonal anti alpha-, beta-, gamma- and delta-sarcoglycan antibodies and/or mouse monoclonal anti beta-, gamma- and delta-sarcoglycan antibodies were located along the muscle plasma membrane, and the cluster formation of different two or three sarcoglycan molecules was observed. The triple immunogold labeling also revealed that the signal of sarcospan molecules are present frequently in doublets and/or triplets with the components of sarcoglycan subcomplex, resulting in the cluster formation of signals of sarcoglycan and sarcospan molecules. The result of this study showed that sarcospan was expressed at the myofiber surface and that sarcospan was present in close association with alpha-, beta-, gamma- and delta-sarcoglycans and formed a functional unit with sarcoglycan subcomplex.