The relative density and distribution of acidic negative residues on the plasma membranes of spermatozoa of seven mammalian species were studied using a colloidal iron hydroxide (CIH) labeling technique. The surface distribution of negative residues is discontinuous on mature spermatozoa from the cauda epididymidis or on ejaculated spermatozoa of the rabbit, mongoose, rat, guinea pig and hamster. Tail regions are generally more densely labeled with CIH than head regions, indicating that sperm tails have more acidic negative charges than heads. In these species, the density of the negative surface charge is rather uniform within a particular segment of the spermatozoa and abruptly changes at the junctions to morphologically distinct segments, suggesting that the biochemical properties of the plasma membrane also change at these junctions. In bull (cauda epididymal) and human (ejaculated) spermatozoa, the entire surfaces of spermatozoa are densely labeled with CIH. Comparison of testicular, caput and cauda epididymal spermatozoa of rabbit, bull and hamster suggests that the negative surface charges of spermatozoa increase with epididymal maturation of spermatozoa. A possible chemical nature of the residue(s) has been suggested from neuraminidase digestion and methylation experiments.