Prosomatostatin is processed into two biologically active peptides, somatostatin (SS)-14 and SS-28. SS-14 is found in many tissues, whereas SS-28 is most prominent in the intestine. Human seminal plasma contained high concentrations (median = 3 nmol/l, range 0.6-76 nmol/l, n = 17) of SS with a higher M(r) than that of SS-28 as determined by gel chromatography. After isolation the SS immunoreactivity was identified as SS-64 based on the N-terminal amino acid sequence Leu-Ala-Glu-Leu-Leu-Ser-Glu-Pro-Asn-Gln-, and M(r) of 7242 determined by m.s. and on reactivity with two antibodies raised against SS-14. SS-64 displaced the binding of [125I-Tyr1]SS-14 to rat cerebrocortical membranes as effectively as SS-14 (IC50 = 1.6 mol/l). The concentration of SS-64 in seminal plasma from vasectomized men was significant higher (P < 0.005) than in normal men (median = 25.4 nmol/l, range 1.5-156 nmol/l, n = 21), suggesting that the synthesis of SS-64 takes place in the male accessory sex glands.