Several methods have been devised for the isolation and labeling of structural components of spermatozoa. Rodent spermatozoa were cleaved rapidly and specifically at the junction of the heads and tails by treatment with various proteases, and the separate components were isolated by density-gradient centrifugation. Treatment with reducing agents released the mitochondrial membranes from the midpiece, exposing the underlying tail structures. Mouse spermatozoa were found to contain about 10(7) sites per cell that bind concanavalin A; most of the sites appear to be on the head, for fluorescein-labeled conjugates of concanavalin A were bound mainly to the acrosomal region. Binding of concanavalin A resulted in rapid agglutination of spermatozoa; mixed agglutinates could be formed with somatic cells, as well as with spermatozoa of other species. Fluorescent probes (naphthalenesulfonic acids) bound to the sperm plasma-membrane and caused an immediate loss of motility. In contrast, ethidium bromide bound to the nuclear structures, but did not cause immediate immobilization. These isolation and probing procedures should facilitate detailed chemical analysis of the major components of mammalian spermatozoa.