The heat shock response as revealed by heat inducible puffing and protein patterns, as well as the localization of the major heat shock genes on the salivary gland polytene chromosomes of the olive fruit fly, Bactrocera oleae (Gmelin), a major agricultural pest, have been studied. After in situ hybridization of specific DNA sequences to the polytene chromosomes of B. oleae, the loci of the major heat shock genes hsp70, hsp83, and a putative hsp68 were detected. The two latter genes gave unique signals, and hsp70 hybridized to two adjacent discrete bands. Eight puffs were induced after heat shock, two of which were found to be the loci of the hsp70 and hsp83 genes. The newly synthesized polypeptides from the salivary glands of B. oleae, after gradual temperature elevation from 25 to 42°C, were analyzed in denaturing acrylamide gels. The most heat inducible polypeptides were found to exhibit relative electrophoretic mobilities of 83, 70, 69, 27, 26, and 23 kDa. The thermotolerance of B. oleae assumed by the above results is discussed in comparison with that observed in Drosophila species.