1α,25-Dihydroxyvitamin D (1,25 D; also known as calcitriol), the hormonal form of vitamin D, can inhibit the proliferation and promote the differentiation of human prostate adenocarcinoma cells. However, little is known about the effects of 1,25 D on the invasive ability of prostate cancer cells. We used an in vitro bioassay of cell invasion (Amgel assay) to examine the effects of 1,25 D and a 'noncalcemic' vitamin D analogue, 1α,25- dihydroxy-16-ene-23-yne-cholecalciferol (16-23-D3), on the invasiveness of three well-characterized human prostate carcinoma cell lines: DU 145, PC-3, and LNCaP. PC-3 and LNCaP cells were poorly invasive in Amgel and were hardly affected by treatment with 1,25 D or 16-23-D3 (<3%). Conversely, DU 145 cells were highly invasive in Amgel, and their invasion was markedly inhibited by 1,25 D and 16,23-D3 (maximally 66 and 59.4%, respectively). This effect was both dose-dependent (doses of 1 x 10-7- x 10-13 M) and time-dependent, with maximal inhibition at 1 x 10-7 M and 72 h. Significant inhibition of invasion was observed at physiological levels of 1,25 D. Neither proliferative indices nor cell cycle kinetics were altered during the experimental exposures. Treatment with 1,25 D and 16-23-D3 caused a selective decrease in the secreted levels of type IV collagenases (MMP-2 and MMP-9). These findings support the hypothesis that 1,25 D reduces the risk of invasive prostate cancer and suggest a role for vitamin D compounds in the chemoprevention of invasive prostate cancer.