We describe the isolation of a cDNA encoding a murine sialic acid-specific 9-O-acetylesterase as well as its expression pattern in cells of both hematopoietic and non-hematopoietic origin. This enzyme catalyzes the removal of O-acetyl ester groups from position 9 of the parent sialic acid N-acetylneuraminic acid. The cDNA is 2105 nt in length and encodes a protein of 541 amino acids with a predicted molecular weight of 61 kDa, not including oligosaccharides linked to eight potential N-glycosylation sites. The cDNA encoding the acetylesterase displays a widespread distribution in various cell lines and tissues. Expression studies of B lineage cell lines and primary fetal liver cells revealed a developmentally regulated expression pattern in cells of hematopoietic origin. Given the importance of 9-O-acetylation of sialic acids, the cloning of the cDNA encoding a sialic acid-specific 9-O-acetylesterase will be helpful in understanding further the regulation of this post-translational modification and the biological consequences thereof.