A line (R-3327-A) of androgen-insensitive squamous cell carcinomas of the prostate of Copenhagen rats has been derived from an established line (R-3327) of androgen-responsive tumors. R-3327-A tumors have a growth rate of approximately 10 times that of R-3327. The response of R-3327-A to hormonal environment has been determined by measuring the growth rate of the tumor transplanted to males, females, and castrated males. No great differences were observed. The metabolism of testosterone by tumor samples was studied following s.c. injections of the labeled steroid. The results show very little 5α-reduction of testosterone compared with that obtained in the prostate gland itself, as well as in the androgen-sensitive R-3327 tumors. A comparison of the presence of 17β-hydroxy-5α-androstan-3-one-binding proteins in cytoplasmic extracts from both lines of tumors shows that the receptor proteins are present only in the androgen-sensitive R-3327 and not in the androgen-insensitive R-3327-A. The levels of the receptor proteins in R-3327 tumors are higher in tumors borne by male than by female animals, and castration of males decreases the amount of 17β-hydroxy-5α-androstan-3-one binding.