An accurate knowledge of tissue optical properties (absorption coefficients, μa, and reduced scattering coefficients, μs') is critical for precise modeling of light propagation in biological tissue, essential for developing diagnostic and therapeutic optical techniques that utilize diffusive photons. A great number of studies have explored the optical properties of various tissue, and these values are not known in detail due to difficulties in the experimental determination and significant variations in tissue constitution. Especially, in situ estimates of the optical properties of brain tissue, a common measurement target in optical imaging, is a challenge because of its layer structure (where the thin gray matter covers the white matter). Here, we report an approach to in situ estimates of the μa and μs' of the gray and white matter in living rat and monkey brains by using femtosecond time-resolved measurements and Monte Carlo simulation. The results demonstrate that the μa of the gray matter is larger than that of the white matter, while there was no significant difference in the μs' between the gray and white matter. The optical properties of the rat brain were very similar to those of the monkey brain except for the μa of the gray matter here.