An 86-kDa heat-shock-protein-encoding (hsp86) cDNA probe permitted to identify, in whole genomic human DNA, two EcoRI fragments of 2.6 and 5.3 kb. These two fragments, as well as an homologous phage lambda VIII1 harboring about 19 kb of human DNA, were isolated from genomic libraries. Sequence analysis revealed that three different genomic hsp86 sequences had been cloned, one of them being the 5' half of a functional gene. This gene contains several introns, as compared to the entire Hsp86-encoding sequence found in lambda VIII1, which represents a processed pseudogene. Cloned hsp86 promoter, with its TATA-box and a heat-shock element upstream at nt positions -25 and -75, respectively, was functional, as verified by fusion to the bacterial chloramphenicol acetyltransferase-encoding gene and its transient expression in vivo. The typical hsp86-type heat-shock regulation was observed, i.e., significant basal activity associated with an inducibility at elevated temperatures. Furthermore, accurate and efficient in vitro transcription was initiated at this hsp86 promoter, resulting in expression of the hsp86 gene, as well as the unrelated sequences.