An acyl-CoA hydrolase, referred to as hBACH, was purified from human brain cytosol, The enzyme had a molecular mass of 100 kDa and 43-kDa subunits, and was highly active with long-chain acyl-CoAs, e,g. a maximal velocity of 295 mu mol/min/mg and K-m of 6.4 mu M for palmitoyl-CoA. Acyl-CoAs with carbon chain lengths of C8-18 were also good substrates, In human brain cytosol, 85% of palmitoyl-CoA hydrolase activity was titrated by an anti-BACH antibody, which accounted for over 75% of the enzyme activity found in the brain tissue. The cDNA isolated for hBACH, when expressed in Escherichia coli, directed the expression of palmitoyl-CoA hydrolase activity and a 44-kDa protein immunoreactive to the anti-EACH antibody, which in turn neutralized the hydrolase activity. The hBACH cDNA encoded a 338-amino acid sequence which was 95% identical to that of a rat homolog. The hBACH gene spanned about 130 kb and comprised 9 exons, and was mapped to 1p36.2 on the cytogenetic ideogram. These findings indicate that the long-chain acyl-CoA hydrolase present in the brain is well conserved between man and the rat, suggesting a conserved role for this enzyme in the mammalian brain, and enabling genetic studies on the functional analysis of acyl-CoA hydrolase.