Clotrimazole is a pharmaceutical used for treatment of fungal infections. It has been found in surface waters outside municipal wastewater treatment plants but data are scarce regarding its effects on aquatic organisms. It is known that clotrimazole and other imidazole fungicides are inhibitors of the enzyme aromatase (CYP 19). Aromatase converts androgens into estrogens and is suggested to be involved in the sex differentiation in amphibians. The aim of the present study was to evaluate effects of larval exposure to clotrimazole on aromatase activity in brain and gonads, and on gonadal differentiation in Xenopus tropicalis frogs. Another purpose was to determine if larval exposure to ethynylestradiol (EE(2)), at a concentration known to cause male-to-female sex reversal, affects aromatase activity in brain and gonads during gonadal differentiation. Tadpoles were exposed from shortly after hatching (Nieuwkoop and Faber developmental stages 47-48) until complete metamorphosis (NF stage 66) to 6, 41, and 375 nM clotrimazole or 100 nM (nominal) EE(2). Aromatase activity was measured in the brain and gonad/kidney complex of tadpoles during gonadal differentiation (NF stage 56) and, in the clotrimazole experiment, also at metamorphosis. In clotrimazole-exposed tadpoles gonadal aromatase activity increased over exposure time in the 41 and 375 nM groups but did not differ significantly from the control group. Gonadal aromatase activity was increased in both sexes exposed to 41 and 375 nM clotrimazole at metamorphosis. Brain aromatase activity was decreased in tadpoles (NF stage 56) exposed to 375 nM clotrimazole, but at metamorphosis no differences were seen between groups or between sexes. No effects of clotrimazole on sex ratio or gonadal histology were noted at completed metamorphosis. EE(2)-exposed tadpoles had a slightly decreased gonadal aromatase activity, though not significantly different from control group, and there was no effect of EE(2) on brain aromatase activity. All EE(2)-exposed tadpoles developed ovaries. These findings indicate that estrogen-induced ovarian differentiation is not paralleled by increased gonadal aromatase activity in X. tropicalis. Further studies are needed, especially on developmental reproductive toxicity, to assess the risk for endocrine disruption in wild amphibians posed by clotrimazole and other imidazole fungicides.