Acetylsalicylic acid (aspirin; ASA) is widely used as an analgesic/antipyretic drug. ASA exhibits a wide range of biological effects, including preventative effects against heart attack, stroke, and the development of some types of cancer. However, the effects of ASA on melanogenesis are not well known. Therefore, we investigated the effect of ASA on melanin production using B16 murine melanoma cells and demonstrated a new biological effect of ASA. In the presence of alpha-melanocyte stimulating hormone (alpha-MSH), B16 melanoma cells are stimulated to enhance melanin synthesis. ASA (2 mM) inhibited alpha-MSH-enhanced melanin synthesis in melanoma more strongly than other well-known anti-melanogenic agents such as arbutin (2 mM) and kojic acid (200 microM). Interestingly, ASA did not inhibit the catalytic activity of mushroom tyrosinase (concentration range 0.5-4.0 mM). To clarify the target of ASA action in melanogenesis, we performed Western blotting for tyrosinase, which is a key melanogenic enzyme. ASA inhibited tyrosinase expression in a dose-dependent manner. Therefore, the depigmenting effect of ASA might be due to inhibition of tyrosinase expression or enhancement of tyrosinase degradation. This study suggests that ASA is a candidate anti-melanogenic agent and it might be effective in hyperpigmentation disorders.