Rapid identification of in vivo affinity ligands would have far-reaching applications for imaging specific molecular targets, in vivo systems imaging, and medical use. We have developed a high-throughput method for identifying and optimizing ligands to map and image biologic targets of interest in vivo. We directly labeled viable phage clones with far-red fluorochromes and comparatively imaged them in vivo by multichannel fluorescence ratio imaging. Using Secreted Protein Acidic and Rich in Cysteine (osteonectin) and vascular cell adhesion molecule-1 as model targets, we show that: 1) fluorescently labeled phage retains target specificity on labeling; 2) in vivo distribution can be quantitated (detection thresholds of approximately 300 phage/mm(3) tissue) throughout the entire depth of the tumor using fluorescent tomographic imaging; and 3) fluorescently labeled phage itself can serve as a replenishable molecular imaging agent. The described method should find widespread application in the rapid in vivo discovery and validation of affinity ligands and, importantly, in the use of fluorochrome-labeled phage clones as in vivo imaging agents.