Deglycosylation of misfolded proteins by the endoplasmic reticulum-associated degradation (ERAD) pathway is catalyzed by peptide:N-glycanases (PNGases) that are highly conserved among mammals and yeast. The catalytic mechanism of PNGases employs a catalytic triad consisting of Cys, His and Asp residues, which is shared by other enzyme families such as cysteine proteases and protein cross-linking transglutaminases (TGases). In contrast to the yeast and mammalian systems, very little is known about ERAD in plants and the enzymes responsible for proper clearance of misfolded plant proteins. We have used a computer-based modeling approach to identify an Arabidopsis thaliana PNGase (AtPNG1). AtPNG1 is encoded by a single-copy gene and displays high structural homology with known PNGases. Importantly, heterologous expression of AtPNG1 restored N-glycanase activity in a PNGase-deficient Saccharomyces cerevisiae mutant. The AtPNG1 gene is uniformly and constitutively expressed at low levels throughout all developmental stages of the plant, and its expression does not appear to be subject to substantial regulation by external stimuli. Recently, recombinant AtPNG1 produced in Escherichia coli was reported to display TGase activity (Della Mea et al., Plant Physiol. 135, 2046-54, 2004). However, inactivation of the AtPNG1 gene did not result in decreased TGase activity in the mutant plant, and recombinant AtPNG1 produced in S. cerevisiae exhibited only residual TGase activity. We propose that the AtPNG1 gene encodes a bona fide peptide:N-glycanase that contributes to ERAD-related protein quality control in plants.