In this study, mixture effects of five dissimilarly acting pesticides were analyzed for antiandrogenic effects in vitro and in vivo. Deltamethrin, methiocarb, prochloraz, simazine, and tribenuron-methyl are all commonly used for agricultural and horticultural purposes. Concentration-response curves for the inhibition of R1881-induced transcriptional activity of the androgen receptor (AR) in vitro of each pesticide alone and in an equimolar mixture were obtained. The IC25 values for deltamethrin, methiocarb, prochloraz, and the mixture were 5.8, 5.8, 3.5, and 7.5 microM, respectively. Simazine and tribenuron-methyl were ineffective. Applying the isobole method resulted in an isobole coefficient of 0.94 at IC25 for the effect of the mixture, indicating additive effects of the compounds. Comparison of observed effects and effects calculated by assuming additivity also strongly indicated additive effects of the pesticides in vitro. In vivo, each of the five pesticides and a mixture of the pesticides were tested for antiandrogenic effects in castrated testosterone-treated Wistar rats. The mixture induced a significant change of weights of the levator ani/bulbocavernosus muscle and adrenal glands. Changes in gene expression in ventral prostates were observed as distinct effects on levels of ornithin decarboxylase (ODC) mRNA and effects on levels of prostate binding protein subunit C3 (PBP C3) mRNA. No pesticide-induced effect on the level of testosterone-repressed prostatic message 2 (TRPM-2) mRNA was observed, whereas flutamide increased TRPM-2 levels. In conclusion, the pesticides were found to act additively in vitro. In vivo, the organ weight changes indicated that the pesticides had an accumulating effect that was not observed for the individual pesticides. Several pesticide-induced gene expression changes were observed, indicating that these are either very sensitive antiandrogenic end-points or that these changes are induced by a pathway not related to AR.