Improved bifunctional chelating agents (BFC) are required for copper-64 radiolabelling of monoclonal antibodies (mAbs) under mild conditions to yield stable, target-specific imaging agents. Four different bifunctional chelating agents (BFC) were evaluated for Fab (Fragment antigen binding) conjugation and radiolabelling with copper-64. Two DOTA- (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and two NOTA- (1,4,7-triazacyclononane-1,4,7-triacetic acid) derivatives bearing a p-benzyl-isothiocyanate group were conjugated to Fab-trastuzumab - which targets the HER2/neu receptor - and the average number of chelators attached ranged from 2.4 to 4.3 macrocycles per Fab. Labelling of the immunoconjugate with copper-64 was achieved in high radiochemical yields after 45 min at 37 °C, and the radiochemical purity of each 64Cu-BFC-Fab-trastuzumab reached 97% after purification. The affinity of each 64Cu-BFC-Fab-trastuzumab ranged between 10 and 50 nM as evaluated by in vitro saturation assays using the HCC1954 breast cancer cell line. PET-MR imaging and biodistribution studies were performed in mice bearing breast cancer BT-474 xenografts. BT-474 tumours were clearly visualized on PET images at 4 and 24 hours post-injection. The tumour uptake of 64Cu-BFC-Fab-trastuzumab reached 8.9 to 12.8% ID g-1 24 hours post-injection and significant differences in non-specific liver uptake were observed depending on the BFC conjugated, the lowest being observed with MANOTA. These results show that MANOTA is a valuable tool for copper-64 radiolabelling.