Two phage clones, lambda hgACL21 and lambda hgACL28, harboring the 5' flanking region of human ATP-citrate lyase (ACL) gene were identified by screening about 1.5 X 10(6) recombinant plaques from the lambdaEMBL3-human placental genomic DNA library. The 5' flanking region of ACL had the CAAT box on -92 bp from the transcription initiation site (+1), however, the TATA box was not found. The primer extension and rapid amplification of cDNA end showed that mRNA is transcribed at a thymine extending 12 bp upstream of the reported cDNA end. The sequences of 5' flanking region in 1.5 kb size of human ACL showed 60% homology with those of rat; however, no homology was found in the exon 1 and intron 1 region. Several consensus sequences, including four Sp1 binding sites, were found in the 5' flanking region of this gene. The promoter activity was assayed by transfecting the 3' or 5' deletion clones of ACL-chloramphenicol acetyl transferase (CAT) plasmid into PLC/PRF5 cells. The clone that contains the part of the first intron sequences from -659 to +440 bp showed the highest CAT activity in the transient transfection assay. High promoter activities were maintained until the transcription initiation site was removed. It is suggested that the sequences from -213 to +12 which contain three Sp1-binding sequences, CAAT box, and the transcription initiation site were necessary as a mean of for exerting the basal promoter activity of ACL gene.