The well-developed thick peritubular matrix (PM) in the middle layer of horse dentin was physico-chemically and also morphologically compared with the intertubular matrix (IM) and the enamel using several microscopes. In scanning laser microscopy, the PM showed a higher laser-beam reflection than the IM, and the contrast image was similar to that of the backscattered electrons by SEM, but to be lower than that of X-ray opacity in microradiography. By SEM-EDX, Ca, P, and Mg were detected from the PM and IM. The mineral content of the PM composed of 15-20 nm sandygrain-shaped crystallites under SEM was significantly higher than that of the IM, but lower than the enamel. The PM/IM in each Ca and P content by weight showed a low average of 1.11, which corresponded to that of their specific gravities previously reported. In Vickers hardness tests, the PM was significantly harder than the IM but softer than the enamel. The PM/IM in the microhardness number showed a high average of 1.85.