In tumor cells, high phosphorylation levels of receptor tyrosine kinases may occur in the absence of exogenous ligands due to autocrine signaling or enhanced tyrosine kinase activity. Here we show that the phosphorylation state of the endogenous epidermal growth factor receptor (EGFR) can be quantitatively imaged in tumor cells and tissues by detecting fluorescence resonance energy transfer between fluorophores conjugated to antibodies against the receptor and phosphotyrosine, respectively. Five different human colorectal cell lines were analyzed for activity and expression of EGFR. All cell lines exhibited basal EGFR phosphorylation under serum starvation conditions. Phosphorylation levels increased after stimulation with EGF or pervanadate, dependent on the level of basal EGFR phosphorylation in the respective cell lines. This basal activity correlated inversely with receptor expression. Using the acceptor photobleaching fluorescence resonance energy transfer imaging approach, a significantly higher phosphorylation state of EGFR was also found in resected human colorectal tumor samples as compared with adjacent healthy tissue. Imaging of EGFR phosphorylation may thus serve as a valuable tool to investigate the role of receptor tyrosine kinase activity in malignant cell growth.