A cDNA encoding human cytosolic acetyl-CoA hydrolase (CACH) was isolated from a human liver cDNA library, sequenced and functionally expressed in insect cells. The human CACH cDNA encodes a 555-amino-acid sequence that is 81.4%/78.7% identical to those of the mouse/rat homologue, suggesting a conserved role for this enzyme in the human and rodent livers. Bioinformatical study further reveals a high degree of similarity among the human and rodent CACHs as follows: First, the gene is composed of 15 exons ranging in size from 56 to 157 bp. Second, the protein consists of two thioesterase regions and a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. Third, the promoter region is GC-rich and contains GC boxes, but lacks both TATA and CCAAT boxes, the typical criteria of housekeeping genes. A consensus peroxisome proliferator responsive element (PPRE) present in the rodent CACH promoter regions supports marked CACH induction in rat liver by peroxisome proliferator (PP).