Translation of a number of mRNAs is under strict regulation via RNA-binding proteins in the spermatogenic cells of testes. A family of Y-box binding proteins represents promising candidates for these presently uncharacterized RNA-binding proteins. The effects of heat stress on the expression of a Y-box binding protein, YB2/RYB-a, and mouse protamine 2 (mP2) were investigated in cultured spermatogenic cells and mouse testes by immunoblot and Northern blot analyses. Localization and alterations in the expression of the YB2/RYB-a protein and the mP2 mRNA in heat-stressed testes were examined by immunohistochemistry and in situ hybridization, respectively. Levels of the YB2/RYB-a protein in spermatogenic cells decreased rapidly as the result of exposure to higher temperature, 37°C or 43°C, compared with the scrotal temperature, 32.5°C, under the culture conditions used. In experimental cryptorchidism, levels of the YB2/RYB-a protein were decreased after Day 10, while the mRNA levels were affected only slightly. The levels of the mP2 mRNA were also decreased and about comparable with those of the YB2/RYB-a protein. Exposure of the lower abdomen to a high temperature, 43°C for 15 min, also damaged the testis and led to a decrease in YB2/RYB-a protein and the mP2 mRNA levels in a coordinated manner. Because YB2/RYB-a is proposed to function as a stabilizer of mP2 mRNA, the perturbation of YB2/RYB-a by heat stress could account for the decline of the mP2 mRNA in elongated spermatids.