To estimate the absolute concentration of substrates surrounding a microdialysis probe in-vivo, we developed a simple calibration method using endogenous glucose as an internal recovery marker and determined the skin distribution of tranilast (N-(3,4-dimethoxy-cinnamoyl)anthranic acid), an anti-allergic agent, in rats. This calibration method was based on the assumption that the concentration of glucose in the extracellular fluid of skin tissues is the same as that in plasma and that the in-vivo recovery ratio of glucose to tranilast by microdialysis is the same as that estimated in-vitro. Based on these assumptions, the dialysate concentrations of tranilast and glucose recovered from cutaneous microdialysis, glucose concentration in plasma, and in-vitro recovery ratio of tranilast to glucose by microdialysis were determined for the estimation of absolute unbound concentration of tranilast in the extracellular fluid of skin tissues. In an in-vitro study employing plasma containing tranilast, the unbound concentration of tranilast in plasma estimated from the dialysate concentration was just comparable with that determined by ultrafiltration methods. Also in an in-vivo study under steady-state plasma concentration of tranilast in rats, the estimated concentration of tranilast in the skin extracellular fluid was the same level as the unbound concentration of tranilast in plasma. Using the present calibration method, the skin distribution of tranilast administered into the intestinal loop or transdermally was continuously monitored in a quantitative manner.