Mushroom tyrosinase has long been used to search its own inhibitor in the cosmetic industry, since this enzyme is easy to obtain and show high enzyme activity. However its structure and catalytic nature are different from those of mouse and human cell lines. In order to provide a correct and convenient tool for screening inhibitors of tyrosinase, tyrosinases from mouse and human melanocytes were obtained from cell culture. Km for L-DOPA as a substrate of mushroom, mouse and human tyrosinases were 0.36 mM, 0.47 mM and 0.42 mM, respectively. Tyrosinase-inhibition patterns by arbutin were competitive in mushroom, noncompetitive in mouse and human. Tyrosinase-inhibition patterns by kojic acid showed mixed type in mushroom, competitive type in mouse, and partial mixed type in human. IC₅₀ of arbutin for mushroom, mouse and human tyrosinases were 85.1 mM, 1.1 mM and 5.8 mM, respectively. IC₅₀ of kojic acid were 7.4 μM, 57.8 μM and 146.2 μM, respectively. Data show that IC₅₀ of arbutin for mouse and human tyrosinases were much lower than that for mushroom tyrosinase, and IC₅₀ of kojic acid for mouse and human tyrosinases were much higher than that for mushroom tyrosinase. Therefore, mushroom tyrosinase is not suitable for the screening and evaluation of tyrosinase inhibitors. On the other hand, melanin biosynthesis in mouse and human cell lines by arbutin and kojic acid were inhibited inversely proportional to IC₅₀.