The gene encoding human carboxyl ester lipase (CEL), including 1628 bp of the 5'-flanking region, has been isolated and characterized from two overlapping lambda phage clones. The gene spans 9832 bp and contains 11 exons interrupted by 10 introns. The exons range in size from 88 to 204 bp, except for the last exon, which is 841 bp. A major and a minor transcription initiation site were determined 13 and 7 bp, respectively, upstream of the initiator methionine. The nucleotide sequence is identical with that of the previously reported cDNA, except for the third nucleotide in the 5'-untranslated sequence, a C, which in the cDNA is a T. A TAAATA sequence is present 26 nt upstream from the major CAP site, and within the 5'-flanking region there are several putative transcription factor binding sites. Seven Alu repetitive sequence elements are present in the region analyzed. The organization of the human CEL gene is similar to that of the recently reported rat pancreatic cholesterol esterase gene. The CEL gene was assigned to chromosome 9q34-qter, which confirms the recently reported results of Tayler et al. (1991, Genomics 10: 425-431). A previously unknown gene with a striking homology to the human CEL gene, here called the CEL-like gene (CELL), has also been isolated and characterized, including 1724 bp of the 5'-flanking region. The CELL gene, which most likely is a psuedogene, spans 4846 bp, and due to the absence of a 4.8-kb segment, the CEL gene exons 2-7 are not present in the CELL gene. Despite these differences, the CELL gene is transcribed. We have also assigned the CELL gene to a separate locus at chromosome 9q34-qter.