Technical Abstract: Sequence-based gene expression data are used to interpret results from functional genomic and proteomics studies. Even though more than 300,000 bovine expressed sequence tags (EST) are available in public databases, a more thorough and directed sampling of the expressed genome is needed to identify new transcripts and improve assembly and annotation of existing transcript sequences. Accordingly, we examined the utility of constructing cDNA libraries synthesized by arbitrarily primed RT-PCR of mRNA from tissues not well represented in the publicly available bovine EST database. A total of 33 cDNA libraries were constructed from healthy and infected mammary gland tissues of Brazilian Gir and Holstein cattle. This series of libraries was used to generate 6,481 open reading frame-expressed sequence tags (ORESTES) that assembled into 1,798 unique sequence elements of which, 1,157 did not significantly match sequence assemblies available in the Bos taurus gene index. However, a total of 264 of these 1,157 sequence elements aligned with mouse and human expressed sequences demonstrating that ORESTES is an effective resource for discovery of novel expressed sequences in cattle. Furthermore, comparison of the alignment position of bovine ORESTES-derived sequence elements to human gene reference sequences suggested that the priming events for cDNA synthesis more often occurred at the central portion of a transcript, which may have contributed to the relatively high rate of novel sequence discovery.