Serum proteins act as weak acids and participate in acid-base balance. Their effects are imprecisely quantified; in particular, the roles of albumin and globulins need reevaluation. We approached the problem in three steps. First, in artificial solutions resembling serum but with human serum albumin as the only protein moiety, we varied the strong ion difference (SID), partial pressure of carbon dioxide (Pco2) and the concentration of albumin [( Alb]) and fixed the concentration of inorganic phosphate [( Pi]). We measured pH and derived the charges on albumin. Second, extending the work of Stewart (Stewart PA. How to understand acid-base. A quantitative acid-base primer for biology and medicine. New York: Elsevier, 1981:1-286), we developed a mathematical model that solves for pH and for the charges on albumin as functions of SID, Pco2, [Pi], and [Alb]. The calculated values fit the observed values well; that is, the model describes well the behavior of these solutions over a wide range of simulated complex acid-base disturbances. Finally, in human serum samples containing both albumin and globulins, we varied SID, Pco2, and total protein concentration [( TP]); we fixed [Pi] and then measured pH and derived the charges on proteins as above. When we applied to these data the computer model developed for albumin alone, the calculated pH and derived charges on albumin values agreed well with the observed pH and derived charges on proteins. We conclude first that human serum globulins play a negligible role in acid-base equilibria, and second, that in normal human serum at pH 7.40 with [TP] = 7 and [Alb] = 4.3 gm/dl, the charges attributed to proteins are approximately 12 mEq/L; this is substantially less than the value of approximately 17 mEq/L given by many contemporary texts, based on work of van Slyke et al. (van Slyke DD, Hastings AB, Hiller A, Sendroy J Jr. Studies of gas and electrolyte equilibria in blood. XIV. Amounts of alkali bound by serum albumin and globulin. J Biol Chem 1928;79:769-80). These findings should be considered when evaluating acid-base balance in patients with abnormal serum albumin concentration, for example, when interpreting values of the anion gap.