In addition to the μ- and δ-opioid receptors previously reported, the SH-SY5Y human neuroblastoma cell line has high levels of κ3 receptors, accounting for 40% of total opioid binding, as measured with [3H]-diprenorphine binding. Competition studies reveal binding profiles for all three receptor classes that are similar to those observed in brain membranes. Differentiation with retinoic acid increases the levels of opioid receptor binding in the cell line, with the largest elevations in κ3 binding. Fully 75% of the increased binding corresponds to κ3 sites, which represent 50% of total opioid receptor binding in differentiated cells. Morphine inhibits forskolin-stimulated cyclic AMP accumulation, and this effect is readily blocked by the μ antagonist d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP). Naloxone benzoylhydrazone, a κ3 agonist, inhibits forskolin-stimulated cyclic AMP accumulation more potently than morphine and is not reversed by CTAP. These studies indicate that SH-SY5Y cells contain high levels of functional κ3 receptors.