Complementary DNA for human cytosolic acetoacetyl-CoA thiolase (CT) was cloned with the use of anti-[human CT] antibody. The human CT cDNA clone (HCT10) has a 1479-bp insert and a 1191-base open reading frame encoding 397 amino acid residues. Partial polypeptide sequences from purified human CT were present in the deduced sequence. In vivo expression analysis showed that HCT10 encoded potassium-ion non-activated acetoacetyl-CoA thiolase with no 3-ketooctanoyl-CoA thiolase activity, which is characteristic for CT. The deduced amino acid sequence has a 34-57% homology with 4 other human thiolases and 4 acetoacetyl-CoA thiolases of microorganisms.