(64)Cu-diacetyl-bis(N(4)-methylthiosemicarbazonate), (64)Cu-ATSM, continues to be investigated clinically as a PET agent both for delineation of tumor hypoxia and as an effective indicator of patient prognosis, but there are still aspects of the mechanism of action that are not fully understood. The retention of radioactivity in tumors after administration of (64)Cu-ATSM in vivo is substantially higher for tumors with a significant hypoxic fraction. This hypoxia-dependent retention is believed to involve the reduction of Cu-ATSM, followed by the loss of copper to cellular copper processing. To shed light on a possible role of copper metabolism in hypoxia targeting, we have compared (64)Cu retention in vitro and in vivo in CaNT and EMT6 cells or cancers after the administration of (64)Cu-ATSM or (64)Cu-acetate. In vivo in mice bearing CaNT or EMT6 tumors, biodistributions and dynamic PET data are broadly similar for (64)Cu-ATSM and (64)Cu-acetate. Copper retention in tumors at 15 min is higher after injection of (64)Cu-acetate than (64)Cu-ATSM, but similar values result at 2 and 16 h for both. Colocalization with hypoxia as measured by EF5 immunohistochemistry is evident for both at 16 h after administration but not at 15 min or 2 h. Interestingly, at 2 h tumor retention for (64)Cu-acetate and (64)Cu-ATSM, although not colocalizing with hypoxia, is reduced by similar amounts by increased tumor oxygenation due to inhalation of increased O2. In vitro, substantially less uptake is observed for (64)Cu-acetate, although this uptake had some hypoxia selectivity. Although (64)Cu-ATSM is stable in mouse serum alone, there is rapid disappearance of intact complex from the blood in vivo and comparable amounts of serum bound activity for both (64)Cu-ATSM and (64)Cu-acetate. That in vivo, in the EMT6 and CaNT tumors studied, the distribution of radiocopper from (64)Cu-ATSM in tumors essentially mirrors that of (64)Cu-acetate suggests that copper metabolism may also play a role in the mechanism of selectivity of Cu-ATSM.