The binding of 15,16,-3H-naltrexone in human, monkey, dog, guinea pig, rat, and mouse plasma was investigated over a range of concentrations, including predicted therapeutic levels. Studies using equilibrium dialysis at 37 degrees indicate that the extent of binding is independent of naltrexone concentration over the concentration range of 1-500 ng/ml for dog plasma and of 0.1-500 ng/ml for human, monkey, guinea pig, rat, and mouse plasma. The extent of naltrexone binding in plasma is similar in the six species studied, the range being from 20% bound in rat plasma to 26% in plasma from beagle and mongrel dogs. This relatively low extent of naltrexone binding in plasma is consistent with previous findings of a large apparent volume of distribution of this drug in the dog. To investigate further the distribution of tritiated naltrexone, the tissue levels of radioactivity in mice at 1, 5, and 15 min after intravenous administration of 8-3H-naltrexone were determined. Naltrexone was rapidly distributed from plasma to tissues, with less than 4% of the dose being present in plasma at 1 min after injection.