Tubular cells have been isolated, characterized and cultured from more than 70 adult cadaver kidneys (postmortem time less than or equal to 12 hr.). Confluent monolayers were observed at 7 days after seeding (10(6) cells/ml.) and cells demonstrating normal human karyotypes have been passaged up to 6 times. Primary isolates and monolayer cultures were negative for Factor VIII activity, and strongly positive for gamma-glutamyltransferase activity and keratin. Ultrastructurally primary isolates consisted of cells with numerous mitochondria, microvilli, cytoplasmic filaments and well-developed endocytotic apparati. Monolayer cultures examined at 7, 14, 21 and 72 days demonstrated less prominent microvilli and the additional structures of desmosomes and cell junctions. Membrane-associated and cytosolic enzyme activities were measured up to 28 days in culture. The membrane-associated enzymes gamma-glutamyltransferase and alkaline phosphatase both exhibited approximately 10-fold decreases in activity during the 1st 7 days in culture. There was an approximately 5-fold increase in pyruvate kinase activity during the same time period, while fructose-1,6-bisphosphatase activity exhibited a 5-fold decrease. Glucose-6-phosphatase activity did not change during the 28 day culture period examined. From 7 to 28 days no further changes were noted in any of the enzyme activities measured. Decreased membrane-associated enzyme activity corresponded to the ultrastructural observation of less prominent microvilli. Increases in glycolytic enzyme activity and decreases in gluconeogenic enzyme activity may reflect the presence of glucose in the culture medium. The morphologic and biochemical evidence suggests that primary isolates and cultures are proximal tubule cells which should provide a well-defined in vitro human system for future studies.