At least five families of mammalian carboxylesterases (CES) catalyse the hydrolysis or transesterification of a wide range of drugs and xenobiotics and may also participate in fatty acyl and cholesterol ester metabolism. In this study, in silico methods were used to predict the amino acid sequences, secondary and tertiary structures, and gene locations for CES3 genes and encoded proteins using data from several mammalian genome projects. Mammalian CES3 genes were located within a CES gene cluster with CES2 and CES6 genes, usually containing 13 exons transcribed on the positive DNA strand. Evidence is reported for duplicated CES3 genes for the chimp and mouse genomes. Mammalian CES3 protein subunits shared 58-97% sequence identity and exhibited sequence alignments and identities for key CES amino acid residues as well as extensive conservation of predicted secondary and tertiary structures with those previously reported for human CES1. The human genome project has previously reported CES3 mRNA isoform expression in several tissues, particularly in colon, trachea and in brain. Predicted human CES3 isoproteins were apparently derived from exon shuffling and are likely to be secreted extracellularly or retained within the cytoplasm. Mouse CES3-like transcripts were localized in specific regions of the mouse brain, including the cerebellum, and may play a role in the detoxification of drugs and xenobiotics in neural tissues and other tissues of the body. Phylogenetic analyses demonstrated the relationships and potential evolutionary origins of the mammalian CES3 family of genes which were related to but distinct from other mammalian CES gene families.