Developing fiber optic probe geometries to selectively measure fluorescence spectra from different sublayers within human epithelial tissues will potentially improve the endogenous fluorescence contrast between neoplastic and nonneoplastic tissues. In this study, two basic fiber optic probe geometries, which are called the variable aperture (VA) and multidistance (MD) approaches, are compared for depth-resolved fluorescence measurements from human cervical epithelial tissues. The VA probe has completely overlapping illumination and collection areas with variable diameters, while the MD probe employs separate illumination and collection fibers with a fixed separation between them. Monte Carlo simulation results show that the total fluorescence detected is significantly higher for the VA probe geometry, while the probing depth is significantly greater for the MD probe geometry. An important observation is that the VA probe is more sensitive to the epithelial layer, while the MD probe is more sensitive to the stromal layer. The effect of other factors, including numerical aperture (NA) and tissue optical properties on the fluorescence measurements with VA and MD probe geometries, are also evaluated. The total fluorescence detected with both probe geometries significantly increases when the fiber NA is changed from 0.22 to 0.37. The sensitivity to different sublayers is found to be strongly dependent on the tissue optical properties. The simulation results are used to design a simple fiber optic probe that combines both the VA and MD geometries to enable fluorescence measurements from the different sublayers within human epithelial tissues. © 2003 Society of Photo-Optical Instrumentation Engineers.