We have analyzed the properties and subcellular localization of synaptophysin (protein p38) in bovine adrenal medulla. In one-dimensional immunoblotting the adrenal antigen appears identical to synaptophysin of rat synaptic vesicles. In two-dimensional immunoblotting it migrates as a heterogeneous band varying in pI from 4.5 to 5.8. Subcellular fractionation by various sucrose gradients revealed that synaptophysin was present in two different cell particles. More than half of the antigens present in adrenal medulla were confined to special membranes that sedimented both with the "large granules" and with microsomal elements. These membranes could be removed from the large granule sediment by washing. In gradients it equilibrated in regions of low sucrose density. These membranes did not contain any markers for chromaffin granules. Less than half of the amount of synaptophysin present in adrenal medulla copurified with chromaffin granules. Despite several variations in the fractionation scheme synaptophysin could not be removed from chromaffin granules. After washing of granule membranes with alkaline solution synaptophysin still cosedimented in gradients with typical granule markers. The concentration of synaptophysin in membranes of chromaffin granules is low (less than 10%) when compared with synaptic vesicles. It is concluded that in adrenal medulla synaptophysin is present in special membranes, probably in high concentration, and in membranes of chromaffin granules, either in a low concentration in all or in a higher concentration in some of them.