The purpose of these studies was to define expression of hsp27 mRNA during the cycle of the seminiferous epithelium and to determine the distribution of hsp27 protein in the rat testis. To study hsp27 mRNA expression, a rat hsp27 cDNA was isolated and sequenced (GenBank no. M86389). The cDNA was used in Northern blot analysis to estimate the relative levels of hsp27 mRNA in rat seminiferous tubule segments selected for different stages of the cycle of the seminiferous epithelium. The level of hsp27 mRNA was low during stages IX-XII of the cycle of the seminiferous epithelium. Approximately 15-fold higher levels of hsp27 mRNA were expressed during stages II-VI, with intermediate levels being expressed during stages XIII-I and VII-VIII. No effect of FSH on hsp27 mRNA expression was detected in cultured Sertoli cells, suggesting that hsp27 synthesis in Sertoli cells is not directly regulated by FSH. The distribution of hsp27 was also studied by use of immunofluorescence in frozen sections of rat testis, in isolated seminiferous tubules, in primary cultures of Sertoli cells isolated from 19-26-day-old rats, in peritubular myoid cells from 26-day-old rats, and in several cell lines. Microfilaments were localized in similar preparations by using rhodamine-phalloidin or BODIPY-phallicidin (Molecular Probes, Eugene, OR). The hsp27 was co-localized with micro-filaments in Sertoli cells from 20-day-old and older rats, but not in Sertoli cells from younger rats. In other cell types, hsp27 was diffusely distributed throughout the cytoplasm. These results demonstrate that hsp27 expression varies with the cycle of the seminiferous epithelium and provide the first direct morphological evidence that hsp27 is associated with microfilaments in a normal, intact tissue. They also suggest that Sertoli cell micro-filaments, by virtue of their associated hsp27, may be different in composition and function from microfilaments of peritubular myoid cells and many other cell types.