Heat shock transcription factors (HSFs) are generally known as regulators of cellular stress response. The mammalian HSF1 functions as a classical stress factor, whereas HSF2 is active during certain developmental processes, including embryogenesis and spermatogenesis. In the present study, we examined HSF2 expression at specific stages of the rat seminiferous epithelial cycle. We found that expression of the alternatively spliced HSF2-alpha and HSF2-beta isoforms is developmentally regulated in a stage-specific manner. Studies on cellular localization demonstrated that HSF2 is present in the nuclei of early pachytene spermatocytes at stages I-IV and in the nuclei of round spermatids at stages V-VIIab. In contrast a strong HSF2 immunoreactivity was detected in small distinct cytoplasmic regions from zygotene spermatocytes to maturation phase spermatids. Immunoelectron microscopic analysis revealed that these structures are mainly cytoplasmic bridges between germ cells. Our results on cellular localization of HSF2 and stage-specific expression of the HSF2 isoforms indicate that HSF2, in addition to its function as a nuclear transcription factor, may be involved in other cellular processes during spermatogenesis, possibly in the sharing process of gene products between the germ cells.