Exponentially growing and stationary phase young and old cultures of the human cell line WI-38 were studied using cytochemistry at the ultrastructural level. 5'-Nucleotidase activity was present in the plasma membrane of all cells examined; in exponentially growing cultures the reaction was more intense in mitotic cells than in interphase cells. An increase in the amount of the reaction product was observed at confluencey, especially in older cells. The reaction of Mg2+-activated adenosine 5'-triphosphatase was absent or very weak in exponentially growing cells and increased at confluency, especially in older cells. Alkaline phosphatase was detectable only in the cell membranes and in intercellular spaces of young cells at confluency. Acid phosphatase activity was increased in old cells, especially at confluency. In these old cells, positive reactions appeared in numerous small lysosomes, autophagic vacuoles and in some flattened sacs of the Golgi apparatus. The obtained results confirm and extend previous biochemical observations and indicate that changes in phosphatase activity are associated with proliferative activity and senescence of cells growing in vitro.