The potential for agricultural chemicals to cause endocrine disruption (ED) in humans and wildlife is an increasing concern; however, the effects of commonly used pesticides at environmentally relevant concentrations are largely unknown. Therefore, 12 environmentally relevant pesticides (11 herbicides and pentachlorophenol (PCP)) were tested for their endocrine disrupting potential in two in vitro assays. A recombinant yeast screen was used to detect receptor mediated (anti-) estrogenic and (anti-) androgenic activity (concentration range: 0.01−1000 μM), and cultured Xenopus oocytes were used to measure effects on the ovulatory response and ovarian steroidogenesis (concentration range: 0.00625−62.5 μM). Eleven pesticides were active in at least one assay (isoproturon, diuron, linuron, 4-chloro-2-methylphenoxy acetic acid (MCPA), mecoprop, atrazine, simazine, PCP, trifluralin, chlorpropham, bentazone), and one had no effect (2,4-dichlorophenoxy acetic acid (2,4,-D)). The most common effects were antiestrogenic/antiandrogenic activity in the yeast screen, and inhibition of ovulation in vitro, accompanied by decreased testosterone production. Estrogenic activity was never observed. In addition, the most potent compound identified in vitro (PCP) was tested for ED activity in vivo. A short-term exposure (6 days) of adult female Xenopus to low concentrations (0.1 or 1 μg/L; 0.375 or 3.75 nM) resulted in minor alterations in plasma hormone levels and toxic effects on the ovary. Changes in in vitro human chorionic gonadotropin (hCG) stimulated hormone production in ovarian follicles from exposed individuals was also observed. In conclusion, novel effects of herbicides and PCP at environmentally relevant concentrations were found, and the effects of these compounds on humans and/or wildlife warrant further investigation.