1 The binding characteristics of the relaxin receptor in rat atria, uterus and cortex were studied using a [33P]-labelled human gene 2 relaxin (B33) and quantitative receptor autoradiography. 2 The binding kinetics of [33P]-human gene 2 relaxin (B33) were investigated in slide-mounted rat atrial sections. The binding achieved equilibrium after 60 min incubation at room temperature (23 ± 1°C) and dissociated slowly. The association and dissociation rate constants were 4.31 ± 0.34 x 108 M-1 min-1 and 1.55 ± 0.38 x 10-3 min-1 respectively. Thus, the kinetic dissociation constant was 3.46 ± 0.59 pM. 3 Binding was saturable to a single population of non-interacting sites throughout atria, in uterine myometrium and the 5th layer of cerebral cortex. The binding affinities (pK(D)) of [33P]-human gene 2 relaxin (B33) were 8.92 ± 0.09 in atrial myocardium and 8.79 ± 0.04 in cerebral cortex of male rats, and 8.79 ± 0.10 in uterine myometrium. Receptor densities in the cerebral cortex and atria were higher than in uterine myometrium, indicating that relaxin also has important roles in nonreproductive tissues. 4 In male rats, treatment with 17β-oestradiol (20μg in 0.1 ml sesame oil s.c., 18-24 h) significantly decreased the density of relaxin receptors in atria and cerebral cortex. Identical treatment in female rats had no significant effect in atria and cerebral cortex, but it significantly increased the density of relaxin receptors in uterine myometrium. 5 Relaxin binding was competitively displaced by porcine and rat native relaxins. Porcine native relaxin binds to the relaxin receptor in male rat atria (8.90 ± 0.02), and cerebral cortex (8.90 ± 0.03) and uterine myometrium (8.89 ± 0.03) with affinities not significantly different from human gene 2 (B33) relaxin. Nevertheless, rat relaxin binds to the receptors with affinities (8.35 ± 0.09 in atria, 8.22 ± 0.07 in cerebral cortex and 8.48 ± 0.06 in uterine myometrium) significantly less than human gene 2 (B33) and porcine relaxins. 6 Quantitative receptor autoradiography is the method of choice for measurement of affinities and densities of relaxin receptor in atria, uterine myometrium and cerebral cortex. High densities were found in all these tissues. 17β-Oestradiol treatment produced complex effects where it increased the densities of relaxin receptors in uterus but decreased those in atria and cerebral cortex of the male rats, and had no effect on the atria and cerebral cortex of the female rats.