Because the most reliable therapy for cancer involves quantitative resection of all diseased tissue, considerable effort has been devoted to improving a surgeon's ability to locate and remove all malignant lesions. With the aid of improved optical imaging equipment, we and others have focused on developing tumor-targeted fluorescent dyes to selectively illuminate cancer nodules during surgery. We describe here the design, synthesis, optical properties, in vitro and in vivo tumor specificity/affinity, pharmacokinetics, preclinical toxicology, and some clinical application of a folate receptor (FR)-targeted NIR dye (OTL38) that concentrates specifically in cancer tissues and clears rapidly from healthy tissues. We demonstrate that OTL38 binds FR-expressing cells with ∼1 nM affinity and eliminates from receptor negative tissues with a half-time of <30 min. We further show that OTL38 enables visualization of malignant lesions at concentrations less than 100-fold those required to elicit signs of toxicity. Since OTL38 also provides excellent tumor contrast in both murine tumor models and human cancer patients, we conclude that OTL38 constitutes an excellent NIR dye for fluorescence-guided resection of malignant lesions in cancer patients.