Transcriptional induction of heat shock genes in response to temperature elevation and other stresses is mediated by heat shock transcription factors (HSFs). Avian cells express two redundant heat-shock responsive factors, HSF1 and HSF3, which differ in their activation kinetics and threshold induction temperature. Unlike the ubiquitous activation of HSF1, the DNA-binding activity of HSF3 is restricted to undifferentiated avian cells and embryonic tissues. Herein we report a reciprocal activation of HSF1 and HSF3 in vivo. Whereas HSF1 mediates transcriptional activity only in the brain upon severe heat shock, HSF3 is exclusively activated in blood cells upon light, moderate and severe heat shock, promoting induction of heat shock genes. Although not activated, HSF1 is expressed in blood cells nuclei in a granular appearance, suggesting regulation of genes other than heat shock genes. Intraspecific comparison of heat sensitive and heat resistant fowl strains indicates that the unique activation pattern of HSF3 in blood tissue is a general phenomenon, non-related to thermal history. Taken together, HSF1 and HSF3 mediate transcriptional activity of adult tissues and differentiated cells in a non-redundant manner. Instead, an exclusive, tissue-specific activation is observed, implying that redundancy may be developmentally related. The physiological and developmental implications are discussed.