Various animal models have been used for studies of bupivacaine cardiovascular toxicity. These studies are difficult to relate to the clinical situation, since the disposition of bupivacaine in the various species is unknown. The serum protein binding of bupivacaine, therefore, was determined in human, sheep, monkey, dog, and rat at physiologicpH using ultrafiltration. Since a mixed acidosis results during a systemic toxicity reaction to bupivacaine, the influences of an acidicpH, resulting from the addition of lactic acid, also was examined. All sera exhibited two classes of binding sites, a high-affinity, low-capacity class (class 1) and a low-affinity, high-capacity class (class 2). When compared to human serum at physiologicpH, a significantly higher (P< 0.05) affinity constant for the class 1 sites was observed for all species studied, with the exception of the rat. All species studied exhibited a significantly lower (P< 0.05) capacity for the class 1 sites. The binding parameters of the class 2 sites displayed no significant difference. An acidpH resulted in a decrease in bupivacaine protein binding over the entire concentration range studied for all species, with the exception of the monkey. Monkey serum exhibited no change in bupivacaine binding with a decrease inpH. Since protein binding explains only a portion of the total disposition of bupivacaine, further delineation of each animal model under both acidotic and physiologic conditions needs to be accomplished before the animal studies currently under investigation can be extrapolated to the clinical situation.