Using a simple whole-cell binding assay, the effect of androgens on receptor binding activity was studied in genital skin fibroblasts (GSF) from normals and patients with either androgen insensitivity syndrome (AIS), isolated micropenis or hypospadias. Expressing the response as a ratio of augmented to basal specific dihydrotestosterone (DHT) binding, there was a 2.4 +/- 0.87 (mean +/- SD) increase following 20 h incubation with 2 nmol/l [3H]-DHT in normal GSF. Mibolerone, a synthetic nonmetabolizable androgen, produced a similar response. Augmentation was temperature dependent, independent of androgen metabolism and suppressed by protein and RNA inhibitors. All patients with isolated micropenis and hypospadias demonstrated normal augmentation of receptor binding activity. There was no response in patients with complete AIS who were receptor negative. In the majority of patients with partial AIS, there was an in vitro response to androgens; those reared as males all virilized with androgen therapy. In contrast, a male patient with partial AIS who failed to respond to high-dose androgen therapy, showed no augmentation of specific androgen binding in GSF preincubated with androgens. This technique may be a useful in vitro bioassay to predict androgen responsiveness in vivo in patients with androgen insensitivity, either soon after birth in the short-term, or later at puberty.