Brain 90- and 100-kDa heat-shock proteins (HSP90 and HSP100) were purified and antibodies against them prepared. The two antibodies were very specific and did not cross-react with each other. In rat, immunoblotting with the anti-HSP90 antibody showed the most abundant presence of HSP90 in testis as well as brain, compared with lung, liver, spleen, kidney, cardiac muscle, ovarium and uterus. The anti-HSP90 antibody showed the presence of a new 105-kDa protein in rat testis. This novel 105-kDa protein was also detected in brain at a very low concentration but not in HeLa cells or other organs including the uterus and ovarium. The testis 105-kDa protein was purified from rat testis; although it was clearly separable from HSP90 by two-dimensional gel electrophoresis, Q-Sepharose and hydroxyapatite column chromatographies, the properties of this protein were very similar to HSP90. The similarity was higher than 60% on peptide mapping with trypsin digestion, the 105-kDa protein cross-reacted with anti-HSP90 antibody, both were bound similarly to heparin-Sepharose gel and both are located in the cytosol fraction. When the 105-kDa protein was fractionated by HPLC, a molecular mass of 195 kDa was calculated, indicating that it is composed of two identical subunits, similarly to HSP90. The 105-kDa protein did not react with the anti-HSP100 antibody. There was a slight similarity between the 105-kDa protein and HSP100 on the peptide mapping. HSP100 was present in the microsomal fraction as well as in the cytosol. It is concluded that the 105-kDa protein is a testis-specific and HSP90-related protein.