Carboxylesterases (CES) are responsible for the detoxification of a wide range of drugs and xenobiotics, and may contribute to cholesterol, fatty acid and lung surfactant metabolism. In this study, in silico methods were used to predict the amino acid sequences, secondary and tertiary structures, and gene locations for horse CES genes and encoded proteins, using data from the recently completed horse genome project. Evidence was obtained for six CES1 genes closely localised on horse chromosome 3, for which the predicted CES1 gene products are > or =74% identical. The horse genome also showed evidence for three other CES gene classes: CES5, located in tandem with the CES1 gene cluster; and CES2 and CES3, located more than 9 million base pairs downstream on chromosome 3. Horse CES2, CES3 and CES5 gene products shared 42-46% identity with each other, and with the CES1 protein subunits. Sequence alignments of these enzymes demonstrated key enzyme and family specific CES protein sequences reported for human CES1, CES2, CES3 and CES5. In addition, predicted secondary and tertiary structures for horse CES1, CES2, CES3 and CES5 subunits showed extensive conservation with human CES1. Phylogenetic analyses demonstrated the relationships and potential evolutionary origins of the horse CES sequences with previously reported sequences for human and other mammalian CES gene products. Several CES1 gene duplication events have apparently occurred following the appearance of the 'dawn' horse approximately 55 million years ago.