Drosophila melanogaster tissues carrying a third chromosome with the deletion Df(3R)Kar(D2) make a 40,000-dalton (Dal) heat shock protein not made by wild type. The unusual polypeptide was inducible in every tissue examined. Tryptic peptide fingerprints showed it to include part of the 70,000-Dal major heat shock protein. Mapping experiments placed the mutation responsible for the 40,000-Dal protein at or close to the kar(D2) deletion. One break point of the deletion is in subdivision 87A, close to or at a heat shock locus that codes for the 70,000-Dal protein. The results are consistent with the possibility that this break point is within a gene for the 70,000-Dal protein, leaving only the initial portion of its coding sequence. This would specify the direction of transcription of the mutant gene as proximal to distal on the normal chromosome. The 87A heat shock locus should contain at least two genes for the 70,000-Dal protein, because embryos homozygous for the kar(D2) deletion and lacking the heat shock locus at 87C, which also codes for the 70,000-Dal protein, nevertheless produced both the 40,000-Dal and the 70,000-Dal proteins upon temperature elevation. Using the presence of the 40,000-Dal protein to monitor chromosome segregation, we found that embryos homozygous for deletions of the heat shock puff site at 93D exhibited a normal electrophoretic pattern of heat shock proteins.