Endogenous progestogens are important regulators of vertebrate reproduction. Synthetic progestins are components of human contraceptive and hormone replacement pharmaceuticals. Both progestogens and progestins enter the environment through a number of sources, and have been shown to cause profound effects on reproductive health in various aquatic vertebrates. Progestins are designed to bind human progesterone receptors, but they also have been shown to strongly activate androgen receptors in fish. Levonorgestrel (LNG) activates fish androgen receptors and induces development of male secondary sex characteristics in females of other species. Although behavior has been postulated to be a sensitive early indicator of exposure to certain environmental contaminants, no such research on the reproductive behavior of gestagen-exposed fish has been conducted to date. The goal of our study was to examine the exposure effects of a human contraceptive progestin, LNG, on the reproductive development and behavior of the viviparous eastern mosquitofish (Gambusia holbrooki). Internal fertilization is a requisite characteristic of viviparous species, and is enabled by an androgen driven elongation of the anal fin into the male gonopodium (i.e., phallus). In this study, we exposed adult mosquitofish to ethanol (EtOH control), 10 ng/L, and 100 ng/L LNG for 8 d using a static replacement exposure design. After 8 d, a subset of males and females from each treatment were examined for differences in the 4:6 anal fin ratio. In addition, paired social interaction trials were performed using individual control males and control females or females treated 10 ng/L or 100 ng/L LNG. Female mosquitofish exposed to LNG were masculinized as evidenced by the elongation of the anal fin rays, a feature normal to males and abnormal to females. LNG caused significant increases in the 4:6 anal fin ratios of female mosquitofish in both the 10 ng/L and 100 ng/L treatments, although these differences were not significant between the two treatments. LNG caused significant increases in the 4:6 anal fin ratio of males exposed to 100 ng/L, with no effects observed in the 10 ng/L treatment. In addition, the reproductive behavior of control males paired with female mosquitofish exposed to 100 ng/L LNG was also altered, for these males spent more time exhibiting no reproductive behavior, had decreased attending behavior, and a lower number of gonopodial thrusts compared to control males paired to control female mosquitofish. Given the rapid effects on both anal fin morphology and behavior observed in this study, the mosquitofish is an excellent sentinel species for the detection of exposure to LNG and likely other 19-nortestosterone derived contraceptive progestins in the environment. (C) 2016 Elsevier Inc. All rights reserved.