The expression of heat shock genes in vertebrates is regulated mainly at the level of transcription by four heat shock transcription factors (HSFs 1-4). Avian cells express at least three HSFs (HSFs 1-3). HSF1 is rapidly activated by even mild heat shock, while HSF3 is activated only by severe heat shock. In contrast, HSF2 is not activated by heat stress and has been speculated to have developmental functions. Here, we examined the temporal and spatial profiles of changes in the levels of these three HSFs in various tissues during chicken development. We found that HSF3 was almost constantly expressed in various tissues during early to late chicken development. The expression of HSF1 was equally high in most tissues early in development and thereafter declined to different levels in a tissue-dependent manner. Thus, HSF3 became the dominant heat-responsive factor in all tissues examined. The magnitude of heat shock response determined by Northern blotting did not always correlate with the level of HSF1 expression, suggesting that not only HSF1 but also HSF3 may be a major factor mediating stress signals to heat shock gene expression in the chicken. In addition, the high-level and ubiquitous expression of HSF2 as well as HSF1 and HSF3 in early embryogenesis suggested the involvement of these factors in all developmental processes.