The relationship between the pH gradient (delta pH) and substrate accumulation was examined in mitochondria from dog renal cortex. Mitochondria were incubated in media containing bicarbonate or nonbicarbonate buffers. Mitochondrial delta pH was at equilibrium after 2 min incubation but citrate accumulation in the matrix space was still increasing. With nonbicarbonate buffer in rotenone-inhibited mitochondria, citrate and alpha-ketoglutarate concentrations in the matrix did not vary between pH 7.5 and 7.1; delta pH decreased from 0.62 to 0.52 as medium pH fell. With decreasing bicarbonate concentration (from 40 to 10 mM) and constant CO2 tension, concentrations of citrate, alpha-ketoglutarate, malate, glutamate, glutamine, and formate increased; pyruvate accumulation was lower at 10 than at 25 mM bicarbonate; delta pH remained constant. When respiratory changes were produced, concentrations of citrate, alpha-ketoglutarate, malate, and glutamate increased as medium pH fell and CO2 tension increased; accumulation of pyruvate, glutamine, and formate was unaffected. delta pH fell from 0.48 to 0.39 as CO2 tension rose from 3 to 12%. In uninhibited mitochondria, 14CO2 formation from labeled citrate was greater with 10 than with 40 mM bicarbonate; this difference as well as the accumulation of citrate in the matrix was blocked by inhibition of the tricarboxylate carrier with 1,2,3-benzenetricarboxylate. These results dissociate effects of acid-base change on mitochondrial substrate accumulation from changes in delta pH. They suggest a direct, bicarbonate-dependent influence of pH on multiple mitochondrial substrate carriers. This phenomenon may play an important role in metabolic regulation in renal cortex.