Acetylesterase and cinnamoyl ester hydrolase activities were demonstrated in culture supernatant of the anaerobic ruminal fungus Neocallimastix patriciarum. A cDNA expression library from N. patriciarum was screened for esterases using beta-naphthyl acetate and a model cinnamoyl ester compound. cDNA clones representing four different esterase genes (bnaA-D) were isolated. None of the enzymes had cinnamoyl ester hydrolase activity, but two of the enzymes (BnaA and BnaC) had acetylxylan esterase activity, bnaA, bnaB and bnaC encode proteins with several distinct domains. Carboxy-terminal repeats in BnaA and BnaC are homologous to protein-docking domains in other enzymes from Neocallimastix species and another anaerobic fungus, a Piromyces sp. The catalytic domains of BnaB and BnaC are members of a recently described family of Ser/His active site hydrolases [Upton, C. & Buckley, J.T. (1995). Trends Biochem Sci 20, 178-179]. BnaB exhibits 40% amino acid identity to a domain of unknown function in the CelE cellulase from Clostridium thermocellum and BnaC exhibits 52% amino acid identity to a domain of unknown function in the XynB xylanase from Ruminococcus flavefaciens. BnaA, whilst exhibiting less than 10% overall amino acid identity to BnaB or BnaC, or to any other known protein, appears to be a member of the same family of hydrolases, having the three universally conserved amino acid sequence motifs. Several other previously described esterases are also shown to be members of this family, including a rhamnogalacturonan acetylesterase from Aspergillus aculeatus. However, none of the other previously described enzymes with acetylxylan esterase activity are members of this family of hydrolases.