The effects of the herbal product Piper methysticum (kava, Kava kava, ‘Awa, Yaqona,) on human P450 isoforms were studied in vitro using both c-DNA-expressed human enzymes and cryopreserved human hepatocytes. Increasing concentrations of an ethanolic extract of dried P. methysticum root and three purified P. methysticum lactones (methysticin, desmethoxyyangonin and yangonin) were tested for their ability to inhibit the catalytic activity of a panel of P450 isoforms (1A2, 2A6, 2C9, C2C19, 2D6, 2E1 and 3A4) present as c-DNA expressed-enzymes and in previously cryopreserved human hepatocytes. In addition, the test compounds’ effect on hepatocyte viability was evaluated by measuring cellular ATP content. In both models, the P. methysticum extract and the three P. methysticum lactones were found to be potent inhibitors of CYPs 1A2, 2C9, 2C19, 2E1 and 3A4 with IC₅₀ values of approximately 10 μM. The test compounds were also moderately cytotoxic to human hepatocytes (EC₅₀ values of approximately 50 μM). Methysticin was the most potent enzyme inhibitor as well as the most cytotoxic, followed by (in order of potency) P. methysticum, root extract, desmethoxyyangonin and yangonin.