Strain and sex differences in μ opioid-mediated antinociception have been reported in rodents. The present studies evaluated μ opioid receptor-mediated G-protein activation in Lewis and Fischer 344 (F344) male and female rats using agonist-stimulated [ 3 5 S]GTPγS binding. Compared to Lewis rats, F344 rats exhibited a 35% higher level of net DAMGO-stimulated [ 3 5 S]GTPγS binding in striatum. Basal [ 3 5 S]GTPγS binding was ~30% lower in thalamus of Lewis than F344 rats. Female Lewis rats also exhibited slightly (~15%) lower basal [ 3 5 S]GTPγS binding in cingulate cortex relative to F344 rats of either sex. The relative efficacies of the μ partial agonists, morphine and buprenorphine, were also examined. Buprenorphine exhibited ~40% lower relative efficacy in the periaqueductal gray in Lewis compared to F344 rats, but no other relative efficacy differences were found between strains or sexes. Moreover, regional differences in the relative efficacy of buprenorphine were also detected in Lewis but not F344 rats. In contrast to these results, the only difference found between sexes was the 13% lower basal [ 3 5 S]GTPγS binding in the cingulate cortex of female compared to male Lewis rats. These results suggest that differences in μ opioid receptor-mediated G-protein activation may contribute to strain differences in opioid antinociception, whereas sex differences may result predominantly from other mechanisms.