The rate of aspirin (acetylsalicylic acid, ASA) deacetylation to salicylate in vivo determines the availability of the intact molecule for therapeutically important transacetylation reactions. Experiments were carried out to assess the relative contribution of a previously isolated human erythrocyte ASA esterase to the overall hydrolysis rate in vivo, as opposed to similar enzymes in other tissues. In vitro, the rates of ASA hydrolysis were relatively slow in plasma from humans and dogs. The hydrolysis rates were significantly greater in whole blood in dogs, rabbits, and humans. In human and canine whole blood, the rate of hydrolysis correlated positively with hematocrit. In vivo studies with dogs showed that ASA decay rates conform to a 2-compartment model. After reduction of hematocrit by a mean of 49% in 4 dogs without previous splenectomy, the "availability" of ASA increased. In 4 dogs with previous splenectomy, this measurement was increased even more after comparable hematocrit reduction. In 2 dogs that underwent a sub-total hepatectomy, ASA availability increased by only 35% and 12.8%, respectively. These results suggest a significant role for the erythrocyte esterase in vivo in the dog and possibly in humans.