This paper presents a phantom which simulates the optical properties of tissue. The phantom absorption coefficient, scattering coefficient, anisotropy factor, and fluorescence quantum yield can be independently varied to investigate the effects of these parameters on fluorescence excitation and emission spectra from 300 to 650 nm. Phantom fluorophores include Flavin Adenine Dinucleotide (FAD) and Rhodamine B. Absorption is controlled by adjusting phantom hemoglobin concentration. On the basis of their smoothly varying scattering coefficient and the relatively low amount of fluorescence contributed to the mixture in comparison to other available scatterers, 1.05-ttm-diameter polystyrene microspheres were selected as a scatterer. Sample inhomogeneities are simulated by preparing the phantom in a gelatin substrate. The optical properties of turbid phantoms determined with the use of indirect techniques agree well with known values as long as μ(1 - g) > μ. Data are presented from dilute, absorbing, and turbid phantoms and inhomogeneous phantoms to qualitatively illustrate the effects of optical properties and sample geometry on fluorescence spectra. The phantom provides the framework for detailed quantitative investigations of the effects of optical properties, sample size, shape, and structure, boundary conditions, and collection geometry on fluorescence spectra. © 1993 Society for Applied Spectroscopy.