The 5' flanking regions of the four small heat shock genes of Drosophila melanogaster from cytological locus 67B have been characterized. Approximately 500 bp of the primary sequence upstream from the proposed site of initiation of translation has been determined and the 5' end of the messenger RNAs have been localized for each gene. Each of the four genes contains an A-T rich sequence, either TATAAATA or TATAAAAG, which is flanked by a G-C rich region. This A-T rich sequence, which ends about 23 bp upstream from the proposed site of initiation of transcription, is similar to those found in most eukaryotic genes. Novel features of these four genes include a region of homology beginning near the proposed site of initiation of transcription and extending about 20 bp into the 5' noncoding region of the genes. This sequence is also found in the gene for the major heat shock protein, hsp 70. The leaders of these five heat shock genes are long, from 111 to 253 bases in length, as well as unusually A rich, from 46% to 51% A. In addition, each of the four small genes contains the sequence ACTTTNA, 195 +/- 12 bp from the proposed site of initiation of transcription.