Ouabain-like factors modulate intracellular Ca2+ concentrations and Ca2+ stores. Recently, a role for Na+-K+-ATPase Na+ transport inhibition as a pivotal event in ouabain signaling was questioned (Kaunitz JD. Am J Physiol Renal Physiol 290: F995-F996, 2006). In the present study, we used a mathematical model of Ca2+ trafficking in cytoplasm and subplasmalemmal microdomains to simulate the pathways through which ouabain can affect Ca2+ signaling: inhibition of active transport by Na+-K+-ATPase alpha1- and alpha2-isoforms, activation of inositol trisphosphate (IP3) production, and increased IP3 receptor (IP3R) conductance. A fundamental prediction is that Na+-K+-ATPase inhibition favors sarcoplasmic reticulum Ca2+ store loading, whereas Src-mediated increases in IP3 production and IP3R sensitization favor store depletion. The model predicts that alpha2-isoform inhibition generates a peak-and-plateau pattern of cytosolic Ca2+ concentration ([Ca2+](cyt)) elevation, whereas alpha1-isoform inhibition yields a monophasic rise. The effects of ouabain-mediated increases in IP3 production or IP3R conductance on [Ca2+](cyt) depend on their relative distributions between cellular microdomains and the bulk cytoplasm. Simulations suggest that the intracellular localization of IP3 production is a pivotal determinant of the changes in compartmental Ca2+ concentrations that can be induced by ouabain. As a consequence of sequestration of the ouabain-sensitive alpha2-isoform into microdomains, inhibition of the alpha2-isoform in rodents is not predicted to significantly affect cytosolic Na+ concentration. Model simulations support the hypothesis that ouabain can enhance agonist-evoked [Ca2+](cyt) transients when its predominant effect is to inhibit alpha2-isoform Na+ transport and, thereby, increase Ca2+ loading into sarcoplasmic reticulum stores.