Atrazine is currently one of the most widely used agricultural pesticides in the US and is the most frequently detected pesticide in ground and surface water. Earlier work by others has raised the possibility that atrazine can act as an endocrine disrupter in rat males. The current study examined testosterone levels following in vivo and in vitro exposure to atrazine. For in vivo exposures, juvenile rat males were administered atrazine 50mg/kg body weight per day by gavage acutely (from postnatal day (pnd) 46 to 48) and chronically (from pnd 22 to 48). In both acutely- and chronically-treated animals, serum and intratesticular levels of testosterone were significantly reduced by approximately 50%. For in vitro exposures, Leydig cells isolated from rats on pnd 49 were co-cultured with 232 microM atrazine in the presence of a maximally stimulating concentration of luteinizing hormone. Compared with cells cultured with vehicle and luteinizing hormone alone, testosterone production by Leydig cells treated with atrazine was reduced by 35%. A similar decrease was observed when Leydig cells were stimulated with dibuterol cAMP (db-cAMP) in lieu of luteinizing hormone, but not when cells were stimulated with hydroxycholesterol, indicating that the effects of atrazine on testosterone production can be bypassed. Taken together, these results demonstrate that atrazine acts as an endocrine disrupter in rat males by directly inhibiting Leydig cell testosterone production.