N-(Dicyclohexyl)acetyl-piperidine-4-benzylidene-4-carboxylic acid (1), although a very potent in vitro 5alpha-steroid reductase (5alphaR) type 2 inhibitor, showed only marginal in vivo activity in rats. Since this could be due to hindered cellular uptake of the carboxylic acid, acid (1) and its corresponding methyl ester (1a) were compared with respect to their permeation properties. In the parallel artificial membrane permeation assay (PAMPA), 1a showed a higher %flux of 55 versus 6 for 1. Considering the high potency of 1 and better permeation of 1a, the use of 1a as a prodrug for 1 was explored using the human prostate carcinoma cell line DU145. Esterase activity, a prerequisite for this prodrug concept was detected employing 4-nitrophenyl acetate (4-NPA) as a substrate. After incubation of DU145 cells with 1 and 1a, respectively, permeated 1a and its hydrolysis to 1 were unequivocally observed by MALDI-TOF MS analyses, whereas 1 could not be detected inside the cells above the detection limit. Regarding biological activity, 1a showed a stronger inhibition of 5alphaR in intact DU145 cells than 1 (IC50 values, 4 microM and > 10 microM for 1a and 1, respectively). These results suggest that the in vivo activity of 1 might be increased by the use of its methyl ester prodrug 1a.