The optical properties (absorption [μ_a], transport scattering [μ′_s] and effective attenuation [μ_eff] coefficients) of normal canine prostate were measured in vivo using interstitial isotropic detectors. Measurements were made at 732 nm before, during and after motexafin lutetium (MLu)–mediated photodynamic therapy (PDT). They were derived by applying the diffusion theory to the in vivo peak fluence rates measured at several distances (3, 6, 9, 12 and 15 mm) from the central axis of a 2.5 cm cylindrical diffusing fiber (CDF). μ_a and μ′_s varied between 0.03–0.58 and 1.0–20 cm⁻¹, respectively. μ_a was proportional to the concentration of MLu. μ_eff varied between 0.33 and 4.9 cm⁻¹ (mean 1.3 ± 1.1 cm⁻¹), corresponding to an optical penetration depth (δ = 1/μ_eff) of 0.5–3 cm (mean 1.3 ± 0.8 cm). The mean light fluence rate at 0.5 cm from the CDF was 126 ± 48 mW/cm² (N = 22) when the total power from the fiber was 375 mW (150 mW/cm). This study showed significant inter- and intraprostatic differences in the optical properties, suggesting that a real-time dosimetry measurement and feedback system for monitoring light fluences during treatment should be advocated for future PDT studies. However, no significant changes were observed before, during and after PDT within a single treatment site.