Maximal expression of the Drosophila heat shock gene hsp70 can be activated by a pair of heat shock consensus elements (HSE's) positioned close to the transcription start site. In contrast, required HSE's of other heat shock genes (i.e., hsp26, 27, 23) are located several hundred base pairs (bp) farther upstream of their start sites. Using germline transformation, we analyzed the requirements for HSE organization in the hsp70 and hsp26 regulatory regions. A 51 bp fragment containing the two proximal hsp70 HSE's was sufficient to rescue the heat shock response of an hsp26-lacZ gene devoid of its HSE's. Heat inducibility was restored with either orientation of the fragment relative to the hsp26 transcription start. In hsp70 gene constructions, relocation of hsp70 HSE's to more remote positions by inserting 127 or 331 bp into the regulatory region failed to substantially reduce expression. Thus, in contrast to their native configurations, the hsp26 promoter can be activated by HSE's solely in a proximal position and the hsp70 promoter can be activated by remote HSE's. In addition, a simple and sensitive assay for quantitative measurement of B-galacto-sidase activity in crude fly extracts is described.