Technical Abstract: Lignin content of switchgrass (Panicum virgatum L.) cell walls is a negative trait for conversion of biomass into liquid fuels. One of the key enzymes in lignin biosynthesis is cinnamyl alcohol dehydrogenase (CAD). In this study, we have shown that CAD activity and protein levels decrease minimally in developmentally distinct switchgrass internodes suggesting that lignification is a continuous process at all stages of tiller development. Transcript abundance indicated that of three CAD/CAD-like sequences indicated PviCAD1 was present in greater abundance than a closely related PviCAD2 sequence. Transcripts for a third CAD-like sequence (PviAroADH) were present at intermediate levels as compared to PviCAD1 and CAD2. 2-dimensional gel electrophoresis indicated that several spots immunologically detected as CAD were found in internode protein extracts. Proteomic analysis indicated the presence of both PviCAD1 and PviCAD2, suggesting both of these CADs are biologically active in switchgrass. We biochemically characterized all three recombinant enzymes. In contrast to PviCAD1 and PviCAD2, the CAD-like PviAroADH appeared to be an enzyme unrelated to lignification based on phylogenetic and protein modeling data. Gene sequence data indicated the existence of a signature motif that appears to distinguish bona-fide CADs from more distantly related alcohol dehydrogenases. To better understand this, we created several CAD mutants using site directed mutagenesis and assayed for activity on aromatic aldehydes and alcohols. We have also cloned CAD from several switchgrass genotypes and have preliminary data on potential CAD-specific SNPs.