The androgen resistance syndromes are generally felt to be due to quantitative or qualitative abnormalities of the androgen receptor. Some patients with testicular feminization have no demonstrable fibroblast cytosol androgen binding, whereas others have androgen binding in cultured fibrobalsts that is thermolabile or fails to be stabilized by sodium molybdate. I describe here familial incomplete testicular feminization associated with reduced nuclear androgen retention. Fibroblasts, cultured from pubic skin biopsies of two phenotypic female 46XY siblings, were assayed for whole cell and nuclear uptake of [(3)H]dihydrotestosterone in dispersed, intact cells. Whole cell binding of [(3)H]dihydrotestosterone at 22 degrees C in the patients' fibroblasts was in the normal range. However, no high affinity, saturable binding of [(3)H]dihydrotestosterone was demonstrable in crude nuclear pellets prepared from the patients' fibroblasts incubated at 37 degrees C with the hormone. Incubating the patients' cells with [(3)H]methyltrienolone or examining the nuclear uptake of [(3)H]dihydrotestosterone in these cells at 22 degrees C did not alter these findings. Although cytosol from the patients' cells revealed a quantitatively diminished 8S peak for [(3)H]dihydrotestosterone after centrifugation on sodium molybdate-containing sucrose gradients, there was no peak of (3)H in the 4S region from 0.3 M KCl nuclear extracts of the patients' cells after they had been incubated with [(3)H]dihydrotestosterone at 37 degrees C. Although whole cell binding studies at 37 degrees C showed minimally diminished androgen binding in the patients' cells compared with binding at 22 degrees C, Griffin (1979. J. Clin. Invest.64: 1624-1631.) has demonstrated thermolability of the androgen receptors in fibroblasts also cultured from these patients. The observations with intact cells coupled with the diminished cytosol 8S peak of [(3)H]dihydrotestosterone on sucrose gradients indicate that these patients have cytosol androgen receptors that are qualitatively abnormal physicochemically, the physiologic consequence of which is failure of nuclear androgen localization. Thus, although the underlying defect in the pathogenesis of the androgen resistance in these patients appears to reside in the androgen receptor, the crucial biologic manifestation of the molecular lesion is impaired nuclear androgen retention. These experiments, therefore, suggest that assessment of nuclear [(3)H]dihydrotestosterone uptake is an effective indicator of the functional integrity of the androgen receptor system in patients with various forms of androgen insensitivity and provides additional insights to those obtained by thermolability or cytosol sucrose gradient studies.