Previously, we reported that acetaminophen (APAP) showed selective toxicity towards melanoma cell lines. In the current study, we investigated further the role of tyrosinase in APAP toxicity in SK-MEL-28 melanoma cells in the presence of a short hairpin RNA (shRNA) plasmid, silencing tyrosinase gene. Results from tyrosinase shRNA experiments showed that APAP led to negligible toxicity in shRNA plasmid-treated cells. It was also found that APAP selectively caused escalation in reactive oxygen species (ROS) formation and intracellular GSH (ICG) depletion in melanocytic human SK-MEL-28 and murine B16-F0 melanoma cells that express functional tyrosinase whereas it lacked significant effects on ROS formation and ICG in amelanotic C32 melanoma cells that do not express functional tyrosinase. These findings suggest that tyrosinase plays a major role in APAP selective induced toxicity in melanocytic melanoma cell lines. Furthermore, the in vivo efficacy and toxicity of APAP in the skin melanoma tumor model in mice was investigated. Mice receiving APAP at 60, 80, 100 and 300 mg/kg/day, day 7 through 13 post melanoma cell inoculation demonstrated tumor size growth inhibition by 7+/-14, 30+/-17, 45+/-11 and 57+/-3%, respectively. Mice receiving APAP day 1 through 13 post melanoma cell inoculation showed tumor size growth inhibition by 11+/-7, 33+/-9, 36+/-20 and 44+/-28%, respectively.