A 6,000-clone metagenomic bacterial artificial chromosome (BAC) library was constructed from microbial flora DNA extracted from the rumen contents of a grass hay-fed dairy cow and activity-based screening was employed to explore the functional hydrolase genes. Ninety four independent clones specifying distinct hydrolytic activities (51 esterases, 18 xylanases and 25 cellulases) were identified. Subcloning and sequence analysis of a subset of these hydrolase-positive clones identified 10 endoglucanase genes. Amino acid sequences of five of these genes indicated less than 55% homology among them, while similarity to the cellulases in the National Center for Biotechnology Information (NCBI) databases averaged 70%. Glycoside hydrolase families 5, 8 and 9 were represented by six, one, and three of the 10 endoglucanases, respectively. Subcloning and sequence analysis of a subset of the esterase-positive clones identified 10 esterase genes. These shared less than 33% homology, with an average similarity of 53% to esterases in the databases, as assessed by predicted amino acid sequence. Preliminary characterization of the encoded cellulases was carried out using crude extracts of each of the subclones. Zymogram analysis using carboxymethylcellulose as a substrate showed a single positive band for each sample, confirming that only one functional cellulase gene was present in each subclone. Optimal pH for these cellulases ranged from 6.5 to 7.0 and their optimal temperatures were 40°C to 50°C. All the endoglucanases could hydrolyze a wide range of β-1,3-, and β-1,4-linked polysaccharides with varying activities. The present work revealed an increased diversity of functional cellulases and esterases in the rumen.