This study examined the effects of protons on cardiac ion channel function in early stages of diabetes mellitus. Transient outward (I(to)) and inward rectifier K+ (IK1) currents were recorded by the whole cell, voltage-clamp technique in ventricular myocytes isolated from hearts of streptozotocin-induced diabetic and control rats. Proton concentration was controlled by independently varying the pH of buffered external or pipette (pHp) solutions. External acidification did not alter I(to) in diabetic rat myocytes when initiated after intracellular dialysis with standard pHp 7.2, but when these cells were dialyzed with acidic pHp (6.6 or 6.0), I(to) density was significantly reduced. Low pHp also reduced I(to) density more in cells from diabetic rats than in controls, whereas alkaline pHp had no effect on either group of cells compared with standard pHp 7.2. In control myocytes dialyzed with pHp 6.0, block of Na+/H+ exchange with 5-(N,N-dimethyl)-amiloride (DMA) or Na(+)-free external solution further reduced I(to) density compared with pHp 6.0 alone, whereas these treatments had less effect on acid-dialyzed cells from diabetic rats. Dialysis with pHp to 6.0 did not alter IK1 in either group of cells compared with standard pHp 7.2, but when done in the presence of DMA or Na(+)-free conditions, IK1 density in both groups was significantly reduced by nearly the same amount. We conclude that intracellular protons inhibit I(to) channels in ventricular myocytes from diabetic and control rats, but that for a given acid load, inhibition is markedly greater in diabetics. This difference may be explained by a diabetes-induced decrease in Na+/H+ exchange that limits proton extrusion during intracellular acidosis. Moreover, acidosis may differentially suppress I(to) and IK1, suggesting that these K+ channels exhibit dissimilar sensitivities to intracellular protons.