Earlier studies have shown that of the four genes (Hsp60A, Hsp60B, Hsp60C, Hsp60D genes) predicted to encode the conserved Hsp60 family chaperones inDrosophila melanogaster, theHsp60A gene (at the 10A polytene region) is expressed in all cell types of the organism and is essential from early embryonic stages, while theHsp60B gene (at 21D region) is expressed only in testis, being essential for sperm individualization. In the present study, we characterized theHsp60C gene (at 25F region), which shows high sequence homology with the other threeHsp60 genes ofD. melanogaster. In situ hybridization of Hsp60C-specific riboprobe shows that expression of this gene begins in late embryonic stages (stage 14 onwards), particularly in the developing tracheal system and salivary glands; during larval and adult stages, it is widely expressed in many cell types but much more strongly in tracheae and in developing and differentiating germ cells. A P-insertion mutant (Hsp60C1) allele with the P transposon inserted at -251 position of theHsp60C gene promoter was generated. This early larval recessive lethal mutation significantly reduces levels ofHsp60C transcripts in developing tracheae and this is associated with a variety of defects in the tracheal system, including lack of liquid clearance. About 10% of the homozygotes survive as weak, shortlived and completely sterile adults. Testes of the surviving mutant males are significantly smaller, with fewer spermatocytes, most of which do not develop beyond the round spermatid stage.In situ and Northern hybridizations show significantly reduced levels of theHsp60C transcripts inHsp60C1 homozygous adult males. The absence of early meiotic stages in theHsp60C1 homozygous testes contrasts with the effect of testis-specificHsp60B (21D) gene, whose mutation affects individualization of sperm bundles later in spermiogenesis. In view of the specific effects in tracheal development and in early stages of spermatogenesis, it is likely that, besides its functions as a chaperone, Hsp60C may have signalling functions and may also be involved in cation transport across the developing tracheal epithelial cells.