Optical technologies, in particular fluorescence spectroscopy, have shown the potential to provide improved detection methods for cervical neoplasia that are sensitive and cost effective through accurate, objective, instantaneous point-of-care diagnostic tools. The specific goals of this study were to analyze reflectance spectra of normal and neoplastic cervical tissue in vivo and to evaluate the data for use in diagnostic algorithm development. Spectroscopic measurements were obtained at four distinct source–detector separations from 324 sites in 161 patients. As the source–detector separation increases, greater tissue depth is probed. The average spectra of each diagnostic class differed at all source–detector separations, with the greatest differences occurring at the smallest source–detector separations. Algorithms, based on principal-component analysis and Mahalanobis distance classification, were developed and evaluated for all combinations of source–detector separations relative to the gold standard of colposcopically directed biopsy. The diagnostic combination of squamous normal versus high-grade squamous intraepithelial lesions gave good discrimination with a sensitivity of 72% and a specificity of 81%; discrimination of columnar normal versus high-grade squamous intraepithelial lesions also was good, with sensitivity of 72% and specificity of 83%. Thus, reflectance spectroscopy appears promising for in vivo detection of cervical precancer. Strategies that combine fluorescence and reflectance spectroscopy may enhance the discrimination capabilities. © 2002 Society of Photo-Optical Instrumentation Engineers.