Changes of 5'-nucleotidase (5'-N) activity during subcultivation of C6 clonal rat glioma cells were studied histochemically, cytochemically and biochemically. Two different cell populations were seeded with high or low cell density respectively. 5'-N activity in cultured cells increased continuously in both populations. The enzyme activity was always higher in the cells growing in high density than in those growing in low density, and this difference became statistically significant after 6 days of subcultivation. It seemed likely that the increase in 5'-N activity depended on cell density, not on the frequency of mitosis. Cytochemically, enzyme activity was detected on the outer surface of the plasma membrane of cell bodies or cell processes. Strong activity was found on the processes and cell membranes where cells touched each other. In the regions where cells had intimate cell-to-cell contact, marked elevation of the enzyme activity was seen. Increased 5'-N activity, which appeared to depend on cell density, was considered to be related with this cell-to-cell contact.