We present structural and comparative analysis of the flanking regions of the rat hsp70.1 stress gene. Several repetitive sequences, microsatellites and short interspersed repetitive elements (SINEs) were found, as well as a significant gap in the 3' UTR, as compared to the orthologous mouse gene. We also show that the complex microsatellite region composed of partially overlapping inverted repeat and long homopurine-homopyrimidine sequence, which is localized 1.8 kbp upstream of the transcription start site, is capable to adopt non-B DNA structures (an H-DNA and a cruciform structure) in vitro. Functional analysis performed with the use of various fragments of the 5'end flanking regions ligated to the chloramphenicol acetyltransferase (CAT) reporter gene revealed a crucial role of cooperation between heat shock element (HSE) regulatory sequences, while none of the three HSEs alone is able to drive efficient heat induced transcription of the reporter gene. We also found that the microsatellite region does not influence transcription by itself, however, it abolishes the effect of the adjacent putative silencing element. To our knowledge, this is a first extensive structural and functional analysis of the promoter region of the mammalian heat inducible hsp70i gene localized distally to the hsp70-related spermatid-specific gene in the major histocompatibility complex III.