The thermoregulated expression of the soybean heat shock (hs) gene Gmhsp17.3-B is regulated via the heat shock promoter elements (HSEs), but full promoter activity requires additional sequences located upstream of the HSE-containing region. Structural features within this putative enhancer region include a run of simple sequences which are also present upstream of HSE-like sequences of other soybean hs genes, and three perfect CCAAT box sequences located immediately upstream from the most distal HSE of the promoter. A series of heterologous and homologous promoter fusions linked to the chloramphenicol acetyl transferase (CAT) gene was constructed and examined in transgenic tobacco plants. The region containing the AT-rich domain of the 5' flanking region was unable to direct transcription from the TATA box of a truncated delta CaMV35S promoter. Heat-inducible CAT activity was detectable when additional sequences from the native promoter containing three CCAAT boxes and a single HSE were present in the constructions. Complete reconstitution of the native hs promoter/enhancer region increased hs specific CAT activities only very little, but deletion of CCAAT box sequences reduced CAT expression five-fold. Our results suggest that AT-rich sequences have a moderate effect on thermoinducible expression levels of the soybean heat shock gene and that CCAAT box sequences act cooperatively with HSEs to increase the hs promoter activity.