Native cellular fluorescence (NCF) represents the innate capacity of tissues to absorb and emit light of a specified wavelength. The ability to define the relationship of in vivo NCF with biological characteristics of neoplastic disease may allow for an improved understanding of the clinical course of disease. Head and neck cancers from 35 patients were evaluated in vivo for NCF characteristics using a xenon lamp-based spectrometer coupled to a handheld fiberoptic probe. Spectral assessment was limited to lambda 450-nm emission characteristics, in which tissues were excited at various wavelengths, ranging from lambda 290 nm to lambda 415 nm, and the intensity of lambda 450 nm emission was recorded. Each cancer was subsequently biopsied and assessed for histological differentiation by a pathologist who was blinded to NCF analysis. Considerable variation in spectral characteristics between head and neck cancers was identified, which was determined, in part, by NCF characteristics of the normal mucosa from the same patient. Poorly differentiated tumors were more likely than well- or moderately differentiated tumors to have lower excitation maxima (P < 0.05 by ANOVA). Most significantly, the tumor differentiation status, as well as the probability of demonstrating recurrent disease, could also be related to the NCF characteristics of the patient's normal mucosa from the same site within the upper aerodigestive tract. NCF analysis may represent an effective tool to identify biological characteristics of head and neck tumors in vivo without the need for invasive biopsies. Results suggest the need to explore the determinants of NCF characteristics expressed by clinically normal mucosa.