There is growing evidence that tonic activity of the opioid system may be important in the modulation of affective state. Naloxone produces a conditioned place aversion in rodents, an effect that is centrally mediated. Previous pharmacological data using antagonists with preferential actions at mu-, delta-, and kappa-opioid receptors indicate the importance of the mu-opioid receptor in mediating this effect. We sought to test the mu-opioid receptor selectivity of naloxone aversion using mu-opioid receptor knock-out mice. mu-Opioid receptor knock-out and wild-type mice were tested for naloxone (10 mg/kg, s.c.) aversion using a place conditioning paradigm. As a positive control for associative learning, knock-out mice were tested for conditioned place aversion to a kappa agonist, U50,488H (2 mg/kg, s.c.). Naloxone produced a significant place aversion in wild-type mice, but failed to have any effect in mu-opioid receptor knock-out mice. On the other hand, both knock-out and wild-type mice treated with U50,488H spent significantly less time in the drug-paired chamber compared to their respective vehicle controls. We conclude that the mu-opioid receptor is crucial for the acquisition of naloxone-induced conditioned place aversion. Furthermore, in a separate experiment using C57BL/6 mice, the delta-selective antagonist naltrindole (10 or 30 mg/kg, s.c.) failed to produce conditioned place aversion.Taken together, these data further support the notion that naloxone produces aversion by antagonizing tonic opioid activity at the mu-opioid receptor.