Patterns of protein synthesis by genital skin fibroblasts from three unrelated normal individuals and three unrelated patients with complete testicular feminization were compared by two-dimensional gel electrophoresis. Cell lines were maintained in monolayer culture and pulse labeled with [35S]methionine. Cells were lysed in 9 M urea, and aliquots of 20 μl subjected to isoelectric focussing and polyacrylamide gel electrophoresis followed by autoradiography. Gels of control fibroblasts showed two proteins (mol wt ~ 45,000, ~ 85,000; pK(i) ~ 5.0) markedly more prominent than on gels from affected fibroblasts. This pattern was unaltered by prior exposure to dihydrotestosterone, suggesting differences in constitutive proteins of the fibroblast cells. Parallel studies demonstrated a marked reduction in the ability of fibroblasts from patients with complete testicular feminization to bind androgens in vitro compared with those of normal individuals. The relationship between these proteins, androgen receptors, and androgen insensitivity requires further investigation.