Aldosterone secretion is enhanced by metabolic acidosis and helps to limit acid-base disturbance by increasing acid excretion by the kidney. One potential target of low plasma pH is the TWIK-related acid sensitive K+ channel (TASK) that is highly expressed in adrenal glomerulosa cells and is inhibited by angiotensin II via the AT1 receptor. Recent studies have shown that mice lacking TASK-1 (K2P3.1) and TASK-3 (K2P9.1) still respond well to acid, and they have enhanced sensitivity to steroidogenic stimuli. Thus, TASK channels are not required for the stimulation of aldosterone production by acid. Therefore, we hypothesized that there may be other acid-sensitive ion channels that are modulated by protons to cause cell depolarization and aldosterone secretion. Adrenal cortical cells from 6-10 day-old rats were isolated by enzymatic digestion and cultured for 24 hr. Extracellular acid (pH 6.0) produced a strong depolarization of 32±5 mV (n=5). Single channel recording showed the presence of weakly inwardly rectifying 36-pS channel in 90% of patches. The 36-pS channels were determined to be TASK-1/3 heteromers based on their single channel conductance, and sensitivities to divalent cations and ruthenium red. In 60% of patches, a 34-pS K+ channel with a high basal activity and distinct biophysical properties was also present. The 34-pS K+ channels were fully inhibited by 1 mM TEA, and more importantly, was inhibited by extracellular acid, with a half maximal inhibition at pH 6.6 (n=4). Therefore, the 34-pS channel is also a target of protons and mediate acid-induced depolarization. The molecular identity of the 34-pS channel is not yet known. Deletion of TASK alone could be compensated by the 34-pS channel activity, and account for the lack of effect of TASK deletion alone on aldosterone secretion. In adrenal cortical cells in the cell-attached configuration, we also recorded a ~30-pS non-selective cation channel that was normally closed but activated by elevation of intracellular [Ca2+] produced by 20 mM KCl or direct application of 20 uM Ca2+ to inside-out patches. As depolarization of adrenal cortical cells augment intracellular [Ca2+] via opening of voltage-dependent Ca2+ channels, we hypothesize a model in which protons inhibit both TASK and the 34-pS K+ channels to depolarize the adrenal cortical cells. As Ca2+ influx is enhanced, the non-selective cation channels are activated to allow additional influx of Na+ and depolarization. We suggest that two acid-sensitive K+ channels and the cation channel participate in acid-induced depolarization and secretion of aldosterone from adrenal cortical cells.