A series of hsp70-phage lambda hybrid genes having various amounts of 5' flanking DNA was introduced into the germ line of Drosophila melanogaster by P-element-mediated transformation. Heat-induced transcription was normal in lines transformed with hsp70-lambda genes having 194 and 146 base pairs of DNA upstream from the mRNA initiation site. Lines transformed with genes having 70 base pairs of upstream DNA accumulated correctly initiated transcripts in response to heat shock, but the amount was somewhat reduced and minor amounts of incorrectly initiated transcripts were observed. No transcription, with or without heat shock, was seen in lines transformed with hsp70-lambda genes having only 52, 44, or 25 base pairs of upstream DNA. Heat shock induced polytene chromosome puffing at the site of integration in a line transformed with a gene having 194 base pairs of upstream DNA. The hsp70-lambda gene and a cotransformed Adh gene closely linked on the same integrating fragment were expressed independently, each apparently responding only to its own normal control signals.