The objective of the study was to assess the epithelial thickness in different lesions of the vocal folds in order to gain more information about its influence on endoscopic imaging. Retrospective study including 161 patients undergoing surgery for a total of 206 benign and malignant lesions of the vocal folds. Laryngoscopy and autofluorescence endoscopy were the first line of investigation for these lesions. Diagnosis was confirmed by histopathology in all cases. Morphometric measurement of epithelial thickness was performed using a normal white light and an autofluorescence microscope. The vocal fold mucosa revealed a progressive thickening from normal epithelium (NE = 147 microm) over the different grades of epithelial dysplasia (EDI = 258 microm, EDII = 301 microm, CIS = 445 microm) to early invasive carcinoma (EIC = 974 microm), while benign lesions presented only a slight epithelial thickening of an additional 100 microm. In such a manner, moderate dysplasia showed a double increase, carcinoma in situ a triple increase and early invasive carcinoma even a sixfold increase of the mean epithelial thickness compared to normal laryngeal mucosa. As fluorescence inducing light has a penetration depth of approximately 300 microm, a marked loss of autofluorescence was recognized from moderate dysplasia onwards, while mild dysplasia simply revealed a slight loss of fluorescence. Autofluorescence microscopy demonstrated a threefold higher fluorescence intensity of the submucosal connective tissue compared to the epithelium, which showed always the same weak intensity independent of the grade of dysplasia. In most cases laryngeal epithelium demonstrates progressive thickening during cancer genesis leading to a loss of autofluorescence.