Synthetic N-formylated peptides are potent chemoattractants for human spermatozoa in vitro. The specific structure-activity relations for eliciting a chemotactic response and the ability of the antagonist tertbutoxycarbonyl-phenylalanyl-leucyl-phenylalanyl-leucyl- phenylalanine (Boc-Phe-Leu-Phe-Leu-Phe) to inhibit the chemotaxis induced by these peptides strongly suggest the presence of receptors on human spermatozoa. The following studies were performed to identify specific binding sites on human spermatozoa by using [35S]-N-formyl-methionyl-leucyl-phenylalanine [( 35S]f-Met-Leu-Phe), a potent chemotactic peptide. Binding of the [35S]formyl-peptide to human spermatozoa was rapid (t1/2, 8 min) and reversible. Binding isotherms of the saturation experiments revealed a single class of high affinity, low capacity binding sites (equilibrium dissociation constant, 17.7 nM; maximal binding, 109 fmol/2 X 10(6) cells) and an average number of 60,000 receptors per cells. The biological potencies of a series of formyl peptides as chemoattractants correlated closely with their relative abilities to compete with [35S]f-Met-Leu-Phe for specific binding to human spermatozoa. These data fulfill the major criteria for demonstration of specific receptors for chemotactic peptides on human spermatozoa. It is likely that these receptor sites initiate the chemotactic response of human spermatozoa to N-formyl peptides.