When hepatocytes are incubated with the chelator diamsar, two pools can be identified, which we have termed extractable and nonextractable. On entering the hepatocyte, 67Cu first associates with the extractable pool and, after approximately 2 h, moves to the nonextractable pool. Both pools demonstrate saturation and are filled as a function of Cu concentration and incubation time. Using the Michaelis-Menten equation, we have estimated the size of the pools after incubation with 67Cu for 30 min and 4 h. During this period the extractable pool decreases in size from 200 +/- 27 to 116 +/- 5 pmol/microgram DNA, whereas the nonextractable pool increases from 28 +/- 9 to 77 +/- 11 pmol/microgram DNA. Movement of Cu from the nonextractable pool to the extractable pool is slow and incomplete. Using [3H]diamsar, we demonstrate that uptake of the chelator is not rate limiting and probably does not occur by pinocytosis. Incubation with diamsar does not affect the activity of superoxide dismutase or cytochrome-c oxidase, although it does prevent the incorporation of 67Cu into ceruloplasmin. Incubation with zinc, which induces metallothionein, results in an increase in 67Cu associated with the nonextractable pool, suggesting that 67Cu-metallothionein constitutes at least part of the nonextractable pool.