Morphine and other μ opioids regulate a number of intracellular signaling pathways, including the one mediated by phospholipase C (PLC). By studying PLC β3-deficient mice, we have established a strong link between PLC and μ opioid-mediated responses at both the behavioral and cellular levels. Mice lacking PLC β3, when compared with the wild type, exhibited up to a 10-fold decrease in the ED 50 value for morphine in producing antinociception. The reduced ED 50 value was unlikely a result of changes in opioid receptor number or affinity because no differences were found in whole-brain B max and K d values for μ, κ, and δ opioid receptors between wild-type and PLC β3-null mice. We also found that opioid regulation of voltage-sensitive Ca 2+ channels in primary sensory neurons (dorsal root ganglion) was different between the two genotypes. Consistent with the behavioral findings, the specific μ agonist [ d -Ala 2 ,(Me)Phe 4 ,Gly(ol) 5 ]enkephalin (DAMGO) induced a greater whole-cell current reduction in a greater proportion of neurons isolated from the PLC β3-null mice than from the wild type. In addition, reconstitution of recombinant PLC protein back into PLC β3-deficient dorsal root ganglion neurons reduced DAMGO responses to those of wild-type neurons. In neurons of both genotypes, activation of protein kinase C with phorbol esters markedly reduced DAMGO-mediated Ca 2+ current reduction. These data demonstrate that PLC β3 constitutes a significant pathway involved in negative modulation of μ opioid responses, perhaps via protein kinase C, and suggests the possibility that differences in opioid sensitivity among individuals could be, in part, because of genetic factors.