The role of local accumulation and diffusion of CO2to modify cellular loss and extracellular accumulation of K+during the initial, reversible phase of myocardial ischemia was investigated in isolated, cylindrical papillary muscles of the rabbit. The muscles were blood-perfused through their vascular tree and placed in a (permanently flowing) humidified gas mixture with predetermined partial pressures of N2, O2, and CO2. Ischemia was produced by total arrest of perfusion and O2withdrawal from the gas mixture. With surface Pco2kept constant during ischemia, [K+]ovaried markedly with muscle geometry. After 10 minutes of ischemia, K+accumulation was ∼2.5 mM in muscles with a radius of 0.35 mm and ∼14 mM in muscles with a radius of 0.9 mm, indicating that a large fraction of K+accumulation was dependent on diffusion of a volatile metabolite. Computer simulation of CO2accumulation and diffusion within a tissue cylinder suggested a close phenomenological relation between Pco2and [K+]oin ischemia. This was confirmed by the finding that an increase of tissue Pco2in small cylinders before or during ischemia by externally applied CO2produced an increase in K+accumulation. The importance of CO2diffusion for local inhomogeneities in K+within the same preparation was demonstrated by showing [K+]ogradients with simultaneous or consecutive measurements between the papillary muscle cylinders and the adjacent septum and within 300 μm from the surface of the papillary muscle cylinders. These gradients predict an inhomogeneity of impulse conduction that might contribute to the genesis of ventricular arrhythmias. Besides the demonstration that accumnulation and diffusion introduce inhomogeneities of [K+]oin ischemia, our results suggest that a significant component of cellular ischemic K+loss is associated with production and extrusion of metabolic acid. On the basis of previous measurements of pHoand pH4in identical conditions, possible mechanisms of ischemic cellular K+loss are discussed.