Polysaccharide-containing vesicles were collected from secretory cells maintained in liquid culture. Characterization of membrane-associated nucleosidephosphatases revealed that the vesicles specifically hydrolyze ATP, have a pH optimum between 6.0 and 6.5, and are stimulated by inorganic cations, especially K(+). The ATPase activity in these vesicles was inhibited by orthovanadate and N,N'-dicyclohexylcarbodiimide; other inhibitors, such as oligomycin, sodium azide, and diethylstilbestrol were generally ineffective. Results from these studies are consistent with the notion that vesicles derived from the Golgi apparatus have partially differentiated into plasmalemma before they fuse with the plasma membrane.