A method was developed for the measurement of the binding of [3H]5α -dihydrotestosterone, [3H]DHT and other steroids at equilibrium with intracellular androgen receptor of genital skin fibroblasts. This method utilized 0.2 M Na2MoO4to stabilize the receptor and Sephadex G-25 chromatography to eliminate steroid metabolism. This binding protein showed the expected limited capacity, high affinity, and specificity of an androgen receptor. Using this method, penile skin cultures from 26 infants with simple hypospadias (HS) were compared with 18 controls. The [3H)DHT binding capacity (Bmax) was 10.1 ± 1.3 (±SE) fmol/mg protein for controls and 6.1 ± 1.7 for HS. The two populations were significantly different by Mann-Whitney test (P< 0.001). Equilibrium dissociation constant was similar for both groups. Surprisingly, there was no correlation between Bmaxand the severity of the anatomic defect. Bmaxwas below the values seen in HS for two of three infants with male pseudohermaphroditism. In complete androgen insensitivity, DHT binding was unmeasurable. A subgroup exists in HS with an abnormality of intracellular androgen receptors. The lack of correlation between severity of hypospadias and Bmaxsuggests that additional factors, such as differences in physicochemical properties of the receptor or factors present in utero, contribute to the development of HS.