1. Pholcodine (3-morpholinoethylmorphine), a semi-synthetic alkaloid, is widely used as an antitussive agent. 2. Norpholcodine [7,8-didehydro-4,5alpha-epoxy-3-(2-morpholinoethoxy)morphinan-6alpha-ol] (NP) and pholcodine-N-oxide [1(9a)-dehydro-(4aR,5S,7aR,9cS,12S)-4a,5,7a,8,9,9a-hexahydro-5-hydroxy-12-methyl-3-morpholinoethoxy-1H-8,9,c-(iminoethano)phenanthro[4,5-bcd] furan-12-oxide] (PNOX) were identified in incubations of pholcodine with freshly isolated rat hepatocytes by liquid chromatography/electrospray-mass spectrometry (LC/ESI-MS). 3. Synthesized NP and PNOX were characterized by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. 4. N-oxidation was the major metabolic pathway for pholcodine, producing a previously unreported metabolite. 5. The metabolism of morphine and codeine was also determined using freshly isolated hepatocytes. 6. For morphine, 3-glucuronidation was the major metabolic pathway, whilst for codeine it was dealkylation (O- and N-). 7. Neither morphine nor its metabolites were metabolites of pholcodine. 8. This observation supports the hypothesis that the absence of analgesic activity with pholcodine may be due to less O-dealkylation in vivo. 9. Together with the slow biotransformation of pholcodine (k(met) = 0.021 microM min(-1)) in comparison with morphine (k(met) = 0.057 microM min(-1)) and codeine (k(met) = 0.112 microM min(-1)), the results obtained were consistent with its low addiction potential and suggest that its antitussive efficacy is mediated by the parent drug or one of its metabolites other than morphine.