Ouabain binds specifically to Na,K-ATPase on the plasma membrane and therefore serves to measure the tissue concentration of Na,K-ATPase. We examined the role of ouabain binding to Na,K-ATPase in its overall tissue distribution. The tissue-to-plasma concentration ratio (Kp,vivo) was defined in each tissue after intravenous administration of 3H-ouabain in guinea pigs, and specific binding of ouabain to Na,K-ATPase was measured in tissue homogenate to obtain the dissociation constant and binding capacity in each tissue. A predicted tissue-to-plasma concentration ratio (Kp,vitro) was calculated using the obtained binding parameters and the volume of extracellular space in each tissue. The absolute values of Kp,vitro were comparable to those of Kp,vivo, except in brain. Regression analysis showed that the specific binding capacity of Na,K-ATPase in each tissue is the main factor in the tissue variation of Kp,vivo. Therefore, the binding of ouabain to Na,K-ATPase plays a significant role in the tissue distribution of ouabain.