In this study, we investigated the interactions of p-fluorofentanyl, an opioid designer drug, fentanyl, sufentanyl, and morphine on cloned human mu-, kappa-, and delta-opioid receptors coexpressed with heteromultimeric G protein-coupled inwardly rectifying K(+) channels (GIRK1/GIRK2) and a regulator of G protein signaling (RGS4) in Xenopus oocytes. We demonstrate that p-fluorofentanyl more potently activates GIRK1/GIRK2 channels through opioid receptors than fentanyl and that the p-fluoro substitution also changes the potency profile from mu > kappa > delta (fentanyl) to mu > delta > or = kappa (p-fluorofentanyl). A comparison of ligand efficacy revealed that morphine, fentanyl, and its analogs less efficiently activate GIRK1/GIRK2 channels through human mu-opioid receptor than [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin. Using site-directed mutagenesis, we investigated whether mutating residues Trp-318 and His-319 to their corresponding residues in kappa- and delta-opioid receptors provides the molecular basis for mu/delta selectivity and mu/kappa selectivity. Changes in EC(50) values for the W318L and W318Y/H319Y mu-opioid receptors show a partial contribution of these residues to the decreased GIRK1/GIRK2 channel activation by fentanyl analogs through kappa- and delta-opioid receptors. The most pronounced effect was observed for p-fluorofentanyl, suggesting that an interaction between the 4-fluorophenylpropanamide moiety of the drug and residues Trp-318 and His-319 is important for the resulting enhanced GIRK1/GIRK2 channel activation through the mu-opioid receptor. Finally, we demonstrate that mutation of W318L confers delta-like potency for morphine on the mutant mu-opioid receptor.