Studies were performed to compare green fluorescent protein (GFP)-transfected and fi re fl y luciferase (Luc)-transfected MCF-7 human breast tumour cells both in vitro and in vivo. For in vitro studies, cells were serially diluted in 96-well microplates and analysed using a NightOwl LB 981 Molecular Light Imager and a Victor multilabel reader. For in vivo studies, nude mice were injected either intraperitoneally, intravenously or subcutaneously with transfected cells and then imaged using the NightOwl Imager after intraperitoneal injection of d-luciferin for Luc tumours, or excitation at 470 nm for GFP tumours. In vitro imaging studies revealed that both GFP and Luc transfectants were quantifiable. However, the Luc-transfected cells were detectable at a significantly lower concentration compared to GFP transfectants. In vivo studies demonstrated that GFP-transfected tumours were detectable as subcutaneous and intraperitoneal tumours but not as deep tissue lesions, whereas Luc-transfected tumours were detectable as subcutaneous and intraperitoneal tumours and as deep tissue lesions resulting from intraperitoneal or intravenous inoculation. These findings demonstrate that GFP-transfected cells may be useful for imaging studies of superficial tumours where both excitation and emission wavelengths are able to penetrate tissues, whereas luciferase-transfected cells appear superior for imaging studies of primary and metastatic tumours in distant sites and deep tissues.