With whole-cell recordings of substantia gelatinosa (SG) neurons from rat spinal cord slices, we investigated the effects of bath application of highly selective delta(1), delta(2), kappa and mu opioid agonists on membrane potential and conductance. Each agonist was applied at 0.5 to 1 micromol/L and evoked robust hyperpolarizations and conductance increases in a subset of neurons. The response magnitude means were similar across agonists at several concentrations; no excitatory effects were observed. Nine of 55 (16%) were hyperpolarized by delta(1) opioids, 2 of 45 (4%) by delta(2), 8 of 59 (14%) by kappa, and 35 of 67 (52%) by mu opioids. To test the hypothesis that SG neurons may be hyperpolarized by multiple opioid subtype agonists, we applied 2, 3, or 4 selective agonists to individual neurons. Most neurons were hyperpolarized only by mu opioids; however, a minority were hyperpolarized by multiple subtype-selective agonists. These results indicate that delta(1)- and delta(2)-selective opioids can also evoke robust hyperpolarizations in spinal SG neurons, that the relative abundance of hyperpolarizing responses was mu > > delta (1) approximately equal kappa > delta(2), and that some SG neurons can be hyperpolarized by more than 1 opioid subtype-selective agonist. These powerful inhibitory postsynaptic responses likely contribute to analgesia evoked by spinally and systemically administered opioid subtype-selective agonists.