Technical Abstract: Tissue-specific cDNA library sequences (expressed sequence tags, or EST) yield a detailed snapshot of gene expression and are useful in developing second-generation molecular resources (i.e. microarrays) for gene expression profiling. The objective of this study was to develop and characterize an intestine-specific cDNA library to examine the transcriptome of the bovine gut and identify expressed genes that influence ruminant nutrition and health. We describe BARC-8BOV, a normalized cDNA library developed from mRNA isolated from four distinct intestinal locations (duodenal, jejunal and ileal small intestine, colon) of Holstein dairy cattle resulting in 19,110 EST deposited into the NCBI Genbank EST database. Assembly and clustering of these 19,110 clone sequences yielded 11,208 unique elements (3,419 contigs and 7,789 singletons) with an average length of 695 base pairs. Analysis strongly suggests normalization and tissue-pooling were effective at increasing the discovery rate of new bovine sequence. A total of 1,123 sequence elements not previously identified in cattle, but with similarity to known genes in other animal species, were identified and shown to be involved in numerous critical biological processes. An additional 745 transcripts were not represented in nucleotide or protein data bases, and further analysis of these could lead to the identification of gut-specific transcript variants of known genes or potentially the discovery of novel bovine genes. Of the 11,208 assembled sequences, 11,034, or 98.4%, match sequences present in the bovine DNA trace archive at NCBI, and add to a bovine EST database previously lacking significant gut tissue representation. Ultimately, these data will also contribute in efforts to annotate the bovine genome.