Human monocyte/macrophage serine esterase (HMSE), commonly known as acid esterase or alpha-naphthylacetate esterase, comprises a group of five enzyme variants that can be distinguished by their isoelectric points from esterase variants of the other normal human blood cell populations. A cDNA for one of the monocytic enzyme variants (HMSE1) was cloned from a U-937 lambda gt11 cDNA library by screening with an oligonucleotide mixture designed according to amino acid sequence data of the purified enzyme. The cDNA contains 1,727 bp with an open reading frame of 1,512 bp coding for a protein of 503 amino acid residues. HMSE1 cDNA represents the first cloned monocyte/macrophage-specific serine esterase and its sequence shows up to 77% homology to other known serine esterases of different species. The amino acid composition of the putative active site of HMSE1 as deduced from the nucleotide sequence corresponds with the active sites of other serine esterases but not with the active sites of serine proteases. Hybridization of the cDNA with RNA of separated normal blood cell populations and hematopoietic cell lines shows restricted expression within the monocyte/macrophage lineage.