The plant-derived steroid, digoxin, a specific inhibitor of Na,K-ATPase, has been used for centuries in the treatment of heart disease. Recent studies demonstrate the presence of a digoxin analog, ouabain, in mammalian tissue, but its biological role has not been elucidated. Here, we show in renal epithelial cells that ouabain, in doses causing only partial Na,K-ATPase inhibition, acts as a biological inducer of regular, low-frequency intracellular calcium ([Ca²⁺]_i) oscillations that elicit activation of the transcription factor, NF-κB. Partial inhibition of Na,K-ATPase using low extracellular K⁺ and depolarization of cells did not have these effects. Incubation of cells in Ca²⁺-free media, inhibition of voltage-gated calcium channels, inositol triphosphate receptor antagonism, and redistribution of actin to a thick layer adjacent to the plasma membrane abolished [Ca²⁺]_i oscillations, indicating that they were caused by a concerted action of inositol triphosphate receptors and capacitative calcium entry via plasma membrane channels. Blockade of ouabain-induced [Ca²⁺]_i oscillations prevented activation of NF-κB. The results demonstrate a new mechanism for steroid signaling via plasma membrane receptors and underline a novel role for the steroid hormone, ouabain, as a physiological inducer of [Ca²⁺]_i oscillations involved in transcriptional regulation in mammalian cells.