Most vital cellular functions are dependent on a fine-tuned regulation of intracellular ion homeostasis. Here we have demonstrated, using COS cells that were untransfected or transfected with wild-type rat ouabain-resistant Na⁺-K⁺-ATPase, that partial inhibition of Na⁺-K⁺-ATPase has a dramatic influence on cell attachment to fibronectin. Ouabain dose-dependently decreased attachment in untransfected cells and in cells expressing wild-type Na⁺-K⁺-ATPase, but not in cells expressing ouabain-insensitive Na⁺-K⁺-ATPase, whereas inhibition of Na⁺-K⁺-ATPase by lowering extracellular K⁺ concentration decreased attachment in all three cell types. Thirty percent inhibition of Na⁺-K⁺-ATPase significantly attenuated attachment. Na⁺-K⁺-ATPase inhibition caused a sustained increase in the intracellular Ca²⁺ concentration that obscured Ca²⁺ transients observed in untreated cells during attachment. Inhibitors of Ca²⁺ transporters significantly decreased attachment, but inhibition of Na⁺/H⁺ exchanger did not. Ouabain reduced focal adhesion kinase autophosphorylation but had no effect on cell surface integrin expression. These results suggest that the level of Na⁺-K⁺-ATPase activity strongly influences cell attachment, possibly by an effect on intracellular Ca²⁺.