The cortical collecting duct (CCD) plays a key role in the regulated K⁺ secretion, which is mediated mainly through ROMK channels located in the apical membrane. However, the mechanisms of the regulation of urinary K⁺ excretion with regards to K⁺ balance are not well known. We took advantage of a recently established mouse CCD cell line (mCCD_cl1) to investigate the regulation of K⁺ secretion by mineralocorticoid and K⁺ concentration. We show that this cell line expresses ROMK mRNA and a barium-sensitive K⁺ conductance in its apical membrane. As this conductance is sensitive to tertiapin-Q ,with an apparent affinity of 6 nM, and to intracellular acidification, it is probably mediated by ROMK. Overnight exposure to 100 nM aldosterone did not significantly change the K⁺ conductance, while it increased the amiloride-sensitive Na⁺ transport. Overnight exposure to a high K⁺ (7 mM) concentration produced a small but significant increase in the apical membrane barium-sensitive K⁺ conductance. The mRNA levels of all ROMK isoforms measured by qRT-PCR were not changed by altering the basolateral K⁺ concentration, but were decreased by 15% to 45% upon treatment with aldosterone (0.3 or 300 nM for 1 and 3 hours). The paradoxical response of ROMK expression to aldosterone could possibly works as a preventative mechanism to avoid excessive K⁺ loss which would otherwise result from the increased electrogenic Na⁺ transport and associated depolarization of the apical membrane in the CCD. In conclusion, mCCD_cl1 cells demonstrate a significant K⁺ secretion, probably mediated by ROMK, which is not stimulated by aldosterone but increased by overnight exposure to a high K⁺ concentration.