Plasma membrane enriched preparations obtained from cultured human skin fibroblasts by differential centrifugation and sucrose density centrifugation techniques were found to contain a Mg2+-dependent Ca2+-stimulated ATPase activity. The specific activity of the (Mg2+ + Ca2+)-ATPase present was 4-5-fold higher than that present in crude membrane preparations and 80-100-fold higher than that present in homogenates. The (Mg2+ + Ca2+)-ATPase activity of both crude and plasma membrane enriched preparations of cultured fibroblasts from cystic fibrosis patients was significantly reduced compared to that activity observed in age-matched controls. This study corroborates our previous observations made in crude homogenate preparations of fibroblasts and indicates another cell type where (Mg2+ + Ca2+)-ATPase activity may be altered in cystic fibrosis.