In a subcellular plasma membrane enriched fraction of bovine corneal epithelium, Ca2+stimulated Mg2+dependent ATPase activity was characterized. This membrane fraction was more than 5-fold and 4-fold enriched with 5′-nucleotidase and alkaline phosphatase activities, respectively, relative to the 100,000×g pellet. With 250μM ATP, maximum stimulation of a high affinity form of Ca2+stimulated Mg2+dependent ATPase activity was obtained with 1.7μM free Ca2+. This activation required no exogenously added Mg2+and was unaffected by either 0.1 mM ouabain, 3μM ruthenium red, 20 mM sodium azide or 0.2μg/ml oligomycin. Exogenous calmodulin (6μM) elicited a 53% increase in this activity which was completely inhibited by 300μM trifluoperazine (TFP). These effects of calmodulin and TFP are consistent with the notion of a plasma membrane origin for this activity and also suggest that this activity could be a basis for the regulation of intracellular Ca2+activity in the submicromolar range