Reuber rat hepatoma cells (R-Y121B) cultured at 0.5% serum accumulated apoalkaline phosphatase in intact cells. When R-Y121B cells were cultured in the presence of bovine serum albumin, alkaline phosphatase activity increased in the cells, and the associated increase in enzyme activity differed amongst bovine serum albumin preparations. The treatment of bovine serum albumin with activated charcoal not only enhanced the effect of serum albumin on alkaline phosphatase activity, but also cancelled the differences due to different preparations of serum albumin. In contrast, no effect from serum albumin was observed in the increase of alkaline phosphatase activity in R-Y121B cell homogenates incubated at 37°C. The activated-charcoal treatment of bovine serum albumin increased the amount of Zn²⁺ bound to the protein. When R-Y121B cells were cultured with bovine serum albumin, the concentration of Zn²⁺ in the cytosol fraction slightly increased. However, the effect of serum albumin on Zn²⁺ concentration in the cytosol fractions was independent of charcoal treatment. It was concluded that serum albumin with Zn²⁺ induces the activation of apoalkaline phosphatase due to Zn²⁺ binding.