The A6 cell line was used to study the role of S-adenosyl-L-homocysteine hydrolase (SAHHase) in the aldosterone-induced activation of the epithelial Na(+) channel (ENaC). Because aldosterone increases methylation of several different molecules, and because this methylation is associated with increased Na(+) reabsorption, we tested the hypothesis that aldosterone increases the expression and activity of SAHHase protein. The rationale for this work is that general methylation may be promoted by activation of SAHHase, the only enzyme known to metabolize SAH, a potent end-product inhibitor of methylation. Although aldosterone increased SAHHase activity, steroid did not affect SAHHase expression. Antisense SAHHase oligonucleotide decreased SAHHase expression and activity. Moreover, this oligonucleotide, as well as a pharmacological inhibitor of SAHHase, decreased aldosterone-induced activity of ENaC via a decrease in ENaC open probability. The kinetics of ENaC in cells treated with antisense plus aldosterone were similar to those reported previously for the channel in the absence of steroid. This is the first report showing that active SAHHase, in part, increases ENaC open probability by reducing the transition rate from open states in response to aldosterone. Thus aldosterone-induced SAHHase activity plays a critical role in shifting ENaC from a gating mode with short open and closed times to one with longer open and closed times.