PAP-1 (5-(4-phenoxybutoxy)psoralen), a potent small-molecule blocker of the voltage-gated potassium Kv1.3 channel, is currently in pre-clinical development for psoriasis. The present study was undertaken to identify the major phase-I metabolites of PAP-1 in rats. Following oral administration at 50 mg/kg, bile, plasma, urine and feces were collected, and separated by reversed-phase HPLC after sample preparation by solid-phase extraction. Five phase-I metabolites, i.e., 5-(oxybutyric-acid)psoralen (M1), 5-[4-(4-hydroxybutoxy)]psoralen (M2), 5-[4-(4-hydroxyphenoxy)]psoralen (M3), 5-[4-(3-hydroxyphenoxy)]psoralen (M4), 8-hydroxyl-5-(4-phenoxybutoxy)psoralen (M5), were isolated and identified by mass spectrometry and NMR spectroscopy. M3, M4 and M5 were hydroxylated products, while M1 and M2 were O-dealkylation products. Incubation of PAP-1 with rat liver microsomes rendered the same five major metabolites in a NADPH-dependent manner suggesting that cytochrome P450 (CYP) enzymes are involved in PAP-1 metabolism. Inhibitors of rat CYP1A1/2 (alpha-naphthoflavone) and CYP3A (ketoconazole) but not CYP2D6, CYP2E or CYP2C9 blocked the metabolism of PAP-1 in rat microsomes. Of the five metabolites M3, M4 and M5 were found to inhibit Kv1.3 currents with nanomolar IC₅₀s, while M1 and M2 were inactive. Our results identified the Kv1.3-inactive M1 as the major phase-I metabolite, and suggest that hydroxylation and O-dealkylation are the major pathways of PAP-1 metabolism principally via CYP1A1/2 and CYP3A. We further conducted a 6-months repeat-dose toxicity study with PAP-1 at 50 mg/kg in both male and female rats and did not observe any changes in hematology, blood chemistry, body weight, histology of any major organ, or speeding up of metabolism suggesting induction of its metabolizing enzymes (presumably CYPs).