The surface of mammalian sperm is known to exhibit regional heterogeneity. Using monoclonal antibodies, we have analyzed the nature of this regional variation at the molecular level. A map of the surface of guinea pig sperm has been constructed that defines a number of regions in which surface antigens are localized and analyzes the diversity of antigens found in each region. In an initial screening of 117 hybridoma antibodies to a sperm membrane preparation, a remarkable result was obtained: all the antibodies bound to a localized region of the cell. From the initial hybrid lines, we established a collection of 56 stable hybridomas producing antibodies to surface antigens. These antibodies detect antigens localized in five surface regions: anterior head (AH), posterior head (PH), whole head (WH), posterior tail (PT), and whole tail (WT). At least 12 distinct surface antigens are recognized that bind antibodies in one of the localized regions (five AH antigens, three PH, two WH, one PT, and one WT). Some of the recognized antigens have been identified as proteins, comprised of either one or several 125I-labeled polypeptides. The identified AH antigens have labeled polypeptides of molecular weights (Mr) 52,000 (52K); 70K, 62K, 46K, 25K, and 18K; 62K, 52K, and 38K; 16K; and 38K. Identified PH antigens have polypeptides of Mr 60K; 66K, 48K, and 41K; and 58K and 48K. Identified WH antigens have polypeptides of Mr 89K and 45K; and 42K. We conclude that the sperm cell can maintain contiguous membrane domains which have quite different compositions. Its surface is a mosaic consisting of multiple regions and each region can contain several localized antigens.