The Na⁺,K⁺-ATPase and the ERK1/2 pathway appear to be linked in some fashion in a variety of cells. The Na⁺,K⁺-ATPase inhibitor ouabain can promote ERK1/2 activation. This involves Src, [Ca²⁺]_i elevation, reactive oxygen species (ROS) generation, and EGF receptor transactivation. In contrast, ERK1/2 can mediate changes in the Na⁺,K⁺-ATPase activity and/or expression. Thus, signaling between ERK1/2 and the Na⁺,K⁺-ATPase can occur from either direction. Whether such bi-directionality can occur within the same cell has not been reported. Here we demonstrate that while ouabain (1 mM) produces only a small (~50%) increase in ERK1/2 phosphorylation in freshly isolated rat salivary (parotid acinar) epithelial cells, it potentiates the phosphorylation of ERK1/2 by sub maximal concentrations of carbachol, a muscarinic receptor ligand that initiates fluid secretion. Although ERK1/2 is only modestly phosphorylated when cells are exposed to 1 mM ouabain or 10^-6 M carbachol, the combination of these agents promotes ERK1/2 phosphorylation to near-maximal levels achieved by a log order carbachol concentration. These effects of ouabain are distinct from Na⁺,K⁺-ATPase inhibition by lowering extracellular K, which promotes a rapid and large increase in ERK1/2 phosphorylation. ERK1/2 potentiation by ouabain (EC₅₀ ~100 µM) involves PKC, Src, and alterations in [Ca²⁺]_i, but not ROS generation or EGF receptor transactivation. In addition, inhibition of ERK1/2 reduces the Na⁺,K⁺-ATPase activity (measured as the stimulation of O₂ consumption by carbachol and the cationophore nystatin). These results suggest that ERK1/2 and the Na⁺,K⁺-ATPase may signal to each other in each direction under defined conditions in a single cell type.