Modified Drosophila heat shock genes were introduced into the germ line by P element transformation. The genes were altered such that several factors could be tested for their influence upon chromosome puffing. Deletion of promoter sequences upstream of position -73 of an hsp7O-lacZ hybrid gene was sufficient to abolish puffing. Analysis of progressive 5′ deletions defines a 16 bp interval that contains sequences required for both heat-induced puffing and gene expression. An internal deletion of the hsp70-lacZ gene that reduces the transcript size from 9 kb to 0.8 kb results in a dramatic reduction in puff size. The chromosomal insertion sites of 26 variant hsp70 or hsp26 genes fall to influence puffing greatly with one marked exception. This transformant possesses an insert that falls to puff and exhibits a tissue-restricted pattern of expression. These results indicate that variation in either promoter strength or transcript length have profound effects on puffing.