To determine whether benzodiazepines (BDZs) quiet isolation distress in 10-day-old rat pups by causing a release of endogenous opioids, a blockade of the effects of chlordiazepoxide (CDP) by the opiate antagonist naltrexone (NLX) was sought. Nonsedating doses of morphine (MOR) (0.125 mg/kg) and CDP (2.0 mg/kg) were equally effective in reducing ultrasonic vocalizations and other indices of isolation distress. Appropriate blocking agents NLX, (0.5 mg/kg) against MOR and Ro 15-1788 (4.0 mg/kg) against CDP returned distress measures to levels of saline-treated rat pups. However, NLX failed to reverse the quieting effects of CDP. If CDP potentiates endogenous opioid release, then NLX should block the CDP effect. A higher dose of CDP did not reveal a release of endogenous opioids, and a higher dose of NLX did not antagonize CDP. The quieting effects of BDZs on isolation distress do not appear to be mediated by the opiate system.