—Arachidonic acid (AA) and other nonesterified fatty acids (FAs) have been shown to exert harmful effects during cardiac ischemia. By continuously measuring intracellular pH (pH_i) changes in neonatal and adult cardiac myocytes, we have found, for the first time, that 10 μmol/L AA induces a substantial intracellular acidosis (0.3 to 0.4 pH units). We have ruled out the possibilities that the AA-induced acidosis is caused by (1) inhibition or stimulation of the pH_i regulators, (2) protein kinase C activation or the generation of AA metabolites or free radicals, or (3) activation of NADPH oxidase or an inward H⁺ current. The AA-induced acidosis fits to a simple diffusion mechanism, as proposed by Kamp and Hamilton (flip-flop model) for artificial phospholipid bilayers. The important properties found in the cardiac myocyte are that (1) the initial rate of acid flux (J_H) increases with the AA concentration (2 to 50 μmol/L), (2) FAs with a ⁻COOH group (eg, AA, oleic acid, and linoleic acid) induce intracellular acidification, but FAs with a ⁻COOCH₃ group (eg, AA methyl ester) have little effect on the pH_i, (3) tetradecylamine (FA amine) induces intracellular alkalosis, and, most importantly, (4) both the AA- and tetradecylamine-induced pH_i changes can be reversed by 0.3% BSA. Because a low concentration of AA (10 μmol/L) can induce a substantial acidosis, the possible involvement of the FA-evoked acidosis in the negative inotropic effect during cardiac ischemia is discussed. The full text of this article is available at http://www.circresaha.org.