The binding characteristics of [3H]oxytocin [( 3H]OT) and [3H]lysine vasopressin [( 3H]LVP) to nonpregnant human myometrium were investigated. Binding of both radioligands was saturable, time dependent, and reversible. Whereas [3H]OT was found to bind to a single class of sites with high affinity [Kd, 1.5 +/- 0.4 (+/- SEM) nM] and low capacity [maximum binding (Bmax), 34 +/- 6 fmol/mg protein], [3H]LVP bound to two classes of sites, one with high affinity (Kd, 2.2 +/- 0.1 nM) and low capacity (Bmax, 198 +/- 7 fmol/mg protein) and another with low affinity (Kd, 655 +/- 209 nM) and high capacity (Bmax, 5794 +/- 1616 fmol/mg protein). The binding of the labeled peptides also displayed a marked difference in sensitivity to Mg2+ and guanine nucleotides. These differences in binding characteristics as well as the differences in potency of analogs in competing for [3H]OT and [3H]LVP binding indicate the presence of distinct receptors for OT and vasopressin in human myometrium. Pharmacological characterization of the high affinity binding sites for [3H]LVP indicated that these are of the V1 subtype. Although, as suggested by others, vasopressin and OT can bind to the same sites, the presence of distinct receptors for both peptides provides an explanation for the previously reported difference in myometrial responsiveness to OT and vasopressin.