The hormone 1alpha,25-dihydroxyvitamin D (1alpha,25(OH)(2)D) inhibits growth and induces differentiation of prostate cells. The enzyme responsible for 1alpha,25(OH)(2)D synthesis, 25-hydroxyvitamin D (25(OH)D)-1alpha-hydroxylase (1alpha-OHase), has been demonstrated in human prostate cells. We compared the levels of 1alpha-OHase activity in prostate cancer cell lines, LNCaP, DU145 and PC-3 and in primary cultures of normal, cancerous and benign prostatic hyperplasia (BPH) prostate cells. We observed a marked decrease in 1alpha-OHase activity in prostate cancer cells, including an undetectable level of activity in LNCaP cells. Transient or stable transfection of 1alpha-OHase cDNA into LNCaP cells increased 1alpha-OHase activity from undetectable to 4.95pmole/mg+/-0.69pmole/mg and 5.8pmole/mg+/-0.7pmole/mg protein per hour, respectively. In response to 25(OH)D, the prohormone of 1alpha,25(OH)(2)D, the transfected LNCaP cells showed a significant inhibition of 3H-thymidine incorporation (37%+/-6% and 56%+/-4% at 10(-8)M for transiently and stably transfected cells, respectively). These findings support an important autocrine role for 1alpha,25(OH)(2)D in the prostate and suggest that the re-introduction of the 1alpha-OHase gene to prostate cancer cells, in conjunction with the systemic administration of 25(OH)D, constitutes an endocrine form of gene therapy that may be less toxic than the systemic administration of 1alpha,25(OH)(2)D.