The objective of these studies was to characterize the relationship between copper levels and Cu/Zn superoxide dismutase (Cu/Zn-SOD) during cellular differentiation. It was hypothesized that the decrease in Cu/Zn-SOD activity that accompanied differentiation would be reversed by supplementing the culture medium with copper. HL-60 cells, a human promyelocytic cell line, were induced to differentiate with retinoic acid and were concurrently supplemented with copper or a copper chelator, tetraethylenepentamine. The results showed that retinoic acid-treated cells contained more copper after differentiation. When the medium was supplemented with copper during retinoic acid treatment, the differentiating cells accumulated more copper than the nondifferentiating cells. Differentiation was accompanied by a significant reduction in Cu/Zn-SOD activity and a slight reduction in Cu/Zn-SOD protein. Activity returned to control values when an extracellular source of copper was provided. Incubation of retinoic acid-treated cells with the chelator showed that they lost proportionally less copper than the noninduced controls. Levels of Cu/Zn-SOD protein were not affected by the copper or chelator treatments. It was concluded that the requirement of differentiating HL-60 cells for copper is not related to providing copper for Cu/Zn-SOD activity. If a supplemental source is not supplied in the medium, then the cells may acquire copper from an intracellular source, namely Cu/Zn-SOD.