Elevated levels of metallothionein (MT) found in rapidly growing tissues such as neonatal liver and various types of human tumors have suggested a role for MT in cell proliferation. To further explore this possibility we investigated the concentration of MT in human colonic cancer (HT-29) cells at different stages of proliferation by means of immunocytochemistry and competitive binding. MT is increased in subconfluent proliferating cells relative to growth-inhibited confluent cells, much as it is in growing tissues. Cycling cells synchronized with compactin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, revealed an oscillation of cytoplasmic MT that reached a maximum in successive late G1 phases and at the G1/S transition. Individual phase of the cell cycle were assessed by [3H]thymidine incorporation and by immunofluorescence employing an antibody that detects a nuclear antigen associated with proliferation. An enzyme-linked immunosorbent assay was used to quantify the relative amounts of MT in homogenate supernatants of HT-29 cells. A 2- to 3-fold increase in MT in actively proliferating cells and the regulation of the protein during the mitotic cell cycle point to a physiological role for MT in cellular proliferation and suggest that it may also serve as a proliferation marker.