We have characterized a mRNA sequence containing the entire coding region of a mouse carboxylesterase (EC 3.1.1.1). pEs-N, an 1840-bp composite of five overlapping cDNA clones, contains an open reading frame of 554 amino acids that display a high degree of similarity with rat and rabbit carboxylesterases. Genetic mapping studies place this carboxylesterase in cluster 1 of the esterase region on chromosome 8. Results of blot hybridization analysis of genomic DNA probed with a pEs-N cDNA under both low and high stringency conditions suggest membership in a carboxylesterase multigene family, as would be expected for a nonspecific carboxylesterase. A message size of 1850-1900 nucleotides was revealed by RNA blot hybridization analysis. S1 nuclease protection analyses with a probe representing a segment of pEs-N detected message in liver, kidney, and lung, but not in spleen, brain, testes, and submandibular gland, with higher levels in female than in male kidney. Additional S1 nuclease-protected mRNA species were found, suggesting the expression of distinct members of a multigene family. In vitro translation of a full-size transcript of pEs-N resulted in a product of 51.5 kDa. Upon the addition of microsomes, this product was processed into a protein of 60.4 kDa, which is within the size range of monomeric units of mouse carboxylesterases.