BACKGROUND & AIMS: Histologic interpretation of disease currently is performed with static images of excised tissues, and is limited by processing artifact, sampling error, and interpretive variability. The aim of this study was to show the use of functional optical imaging of viable mucosa for quantitative evaluation of colonic neoplasia in real time. METHODS: Fluorescein (5 mg/mL) was administered topically in 54 human subjects undergoing screening colonoscopy. Fluorescence images were collected with 488-nm excitation at 12 frames/s with the confocal microendoscopy system. Movement of fluorescein in the transient period (<5 s) and the lamina propria:crypt contrast ratio in the steady-state phase (>5 s) were quantified. RESULTS: Normal mucosa showed circular crypts with uniform size, hyperplasia revealed proliferative glands with serrated lumens, and adenomas displayed distorted elongated glands. For t less than 5 seconds, fluorescein passed through normal epithelium with a peak speed of 1.14 +/- 0.09 microm/s at t = 0.5 seconds, and accumulated into lamina propria as points of fluorescence that moved through the interglandular space with an average speed of 41.7 +/- 3.4 microm/s. Passage of fluorescein through adenomatous mucosa was delayed substantially. For t greater than 5 seconds, high sensitivity, specificity, and accuracy was achieved using a discriminant function to evaluate the contrast ratio to distinguish normal from lesional mucosa (91%, 87%, and 89%, respectively; P < .001), hyperplasia from adenoma (97%, 96%, and 96%, respectively; P < .001), and tubular from villous adenoma (100%, 92%, and 93%, respectively; P < .001). CONCLUSIONS: Confocal imaging can be performed in vivo to assess the functional behavior of tissue in real time for providing pathologic interpretation, representing a new method for histologic evaluation.