Explicit dosimetry of photodynamic therapy requires detailed knowledge of the light, drug, and oxygenation distributions within the target tissue. We present a method for the optical detection and three-dimensional reconstruction of hemoglobin concentration and oxygenation and sensitizer concentration within the human prostate. Spectrally resolved diffuse transmission measurements were made using a small isotropic fiber-based white light source and an isotropic detector inserted into the prostate via parallel closed transparent catheters. The spectra were modeled using the diffusion approximation appropriate for infinite media. The optical absorption of the prostate was assumed to be a linear combination of the absorption spectra of oxy- and deoxyhemoglobin and MLu, and the scattering was assumed to be of the form A(λ/λ). The separation of absorption and scattering coefficients was accomplished based on the spectral shape of the diffuse transmission, rather than the spatial variation in intensity. By making multiple measurements at various source-detector separations, we investigate the signal-to-noise sensitivity of our algorithm. In addition, the redundancy in our source-detector position matrix creates several positions in which the tissue parameters can be reconstructed from multiple independent measurements, allowing an assessment of the repeatability of the algorithm. We find significant heterogeneity in the reconstructed optical properties; however the recovery of spectrally consistent absorption and scattering spectra is improved compared to wavelength-wise reconstruction algorithms.