AIMS: Interindividual differences in analgesic drug response complicate the clinical management of pain. We aimed to identify genetic factors responsible for variable sensitivity to analgesic drugs of disparate neurochemical classes. METHODS AND RESULTS: Quantitative trait locus mapping in 872 (C57BL/6x129P3)F2 mice was used to identify genetic factors contributing to variability in the analgesic effect of opioid (morphine), alpha2-adrenergic (clonidine), and cannabinoid (WIN55,212-2) drugs against thermal nociception. A region on distal chromosome 1 showing significant linkage to analgesia from all three drugs was identified. Computational (in silico) genetic analysis of analgesic responses measured in a panel of inbred strains identified a haplotype block within this region containing the Kcnj9 and Kcnj10 genes, encoding the Kir3.3 (GIRK3) and Kir4.1 inwardly rectifying potassium channel subunits. The genes are differentially expressed in the midbrain periaqueductal gray of 129P3 versus C57BL/6 mice, owing to cis-acting genetic elements. The potential role of Kcnj9 was confirmed by the demonstration that knockout mice have attenuated analgesic responses. CONCLUSION: A single locus is partially responsible for the genetic mediation of pain inhibition, and genetic variation associated with the potassium channel gene, Kcnj9, is a prime candidate for explaining the variable response to these analgesic drugs.