In the present study, Xenopus laevis tadpoles were chronically exposed to four concentrations of the synthetic gestagen Levonorgestrel (LNG; 10−11, 10−10, 10−9, and 10−8M) starting at Nieuwkoop and Faber (NF) stage 48 until completion of metamorphosis. At NF 58 and 66, brain-pituitary and gonad samples were taken for gene expression analyses of gonadotropins and gonadal steroidogenic enzymes. Exposure to 10−9 and 10−8M LNG until NF 58 repressed messenger RNA (mRNA) expression of luteinizing hormone (LH) β in both genders. This decrease was persistent after further treatment until NF 66 in the 10−8M LNG treatment. Expression of follicle-stimulating hormone (FSH) β was affected sex-specifically. No effect was present in NF 58 females, whereas LNG at 10−9 and 10−8M significantly increased FSHβ mRNA levels in males. In NF 66 females, 10−9M LNG treatment increased FSHβ gene expression, whereas a decrease was observed in NF 66 males exposed to 10−8M LNG. In gonads, expression of steroid-5-alpha-reductase was affected sex-specifically with increased mRNA levels in females but repressed levels in males. Gene expression of further gonadal steroidogenic factors was decreased by 10−8M LNG in both genders at NF 66. Assessment of gonad gross morphology and histology revealed poorly developed testes in the 10−8M LNG treatment. Our results reveal considerable effects of chronic LNG exposure on sexual development of amphibians. The persistent inhibition of LHβ expression concomitant with decreased mRNA levels of gonadal steroidogenic enzymes is suggested to result in the disruption of reproduction in adult amphibians.