The aim of this study was to evaluate in vitro and in vivo imaging of HER-2-over-expressing tumours using near-infrared optical imaging. A fluorochrome probe was designed by coupling Cy5.5 to anti-HER-2 antibodies. Cells over-expressing (SK-BR-3 cells) or normally expressing (PE/CA-PJ34 cells) the HER-2 protein were incubated with the probe. After removing unbound probe molecules, fluorescence intensities were determined (a.u.: arbitrary units). Cells were additionally investigated using FACS and laser scanning microscopy. The probe was also injected intravenously into tumours bearing SK-BR-3 ( n=3) or PE/CA-PJ34 ( n=3). Whole-body fluorescence images were generated and analysed. The incubation of SK-BR-3 cells with the probe led to higher fluorescence intensities [2,133 (+/-143) a.u.] compared to controls [975 (+/-95) a.u.]. The results from FACS and immunocytochemical analysis were in agreement with these findings. A distinct dependency between the fluorescence intensity and the cell number used in the incubations was detected. In vivo, the relative fluorescence intensities in SK-BR-3 tumours were higher than in PE/CA-PJ34 tumours at 16-24 h after probe application. HER-2-over-expressing tumours were depictable in their original size. Labelling of HER-2 with Cy5.5 is suitable for in vitro and in vivo detection of HER-2-over-expressing tumour cells.