Cultures from various normal and neoplastic cell lines exfoliated vesicles with 5'-nucleotidase activity which reflected the ecto-enzyme activity of the parent monolayer culture. The ratio of 5'-nucleotidase to ATPase activity in the microvesicles indicated that cellular ecto-ATPase was conserved in the exfoliative process. Phospholipids of the microvesicles contained significantly increased amounts of sphingomyelin and total polyunsaturated fatty acids. It was concluded that the shedded vesicles constituted a select portion of the plasma membrane. Examination by electron microscopy showed the vesicles had an average diameter of 500 to 1000 nm and often contained a second population of vesicles about 40 nm in diameter. As much as 70% of the plasma membrane ecto-5'-nucleotidase activity of a culture was released into the medium over a 24-h period. Phosphoesterhydrolases from C-6 glioma or N-18 neuroblastoma microvesicles dephosphorylated cell surface constituents when in contact with monolayer cultures. Exfoliated membrane vesicles may serve a physiologic function; it is proposed that they be referred to as exosomes.