Gene targeting was used to delete exon 2 of mouse DOR-1, which encodes the δ opioid receptor. Essentially all 3H-[D-Pen²,D-Pen⁵]enkephalin (³H-DPDPE) and 3H-[D-Ala²,D-Glu⁴]deltorphin (³H-deltorphin-2) binding is absent from mutant mice, demonstrating that DOR-1 encodes both δ₁ and δ₂ receptor subtypes. Homozygous mutant mice display markedly reduced spinal δ analgesia, but peptide δ agonists retain supraspinal analgesic potency that is only partially antagonized by naltrindole. Retained DPDPE analgesia is also demonstrated upon formalin testing, while the nonpeptide δ agonist BW373U69 exhibits enhanced activity in DOR-1 mutant mice. Together, these findings suggest the existence of a second delta-like analgesic system. FinallyDOR-1 mutant mice do not develop analgesic tolerance to morphine, genetically demonstrating a central role for DOR-1 in this process.