Vitamin B(6) (pyridoxine and its vitamers) plays an essential role as a co-factor for enzymatic reactions and has also recently been implicated in defense against cellular oxidative stress. The biosynthetic pathway was thoroughly characterized in Escherichia coli, however most organisms, including plants, utilize an alternate pathway involving two genes, PDX1 and PDX2. Arabidopsis thaliana contains one copy of PDX2, but three full-length copies of PDX1, one each on chromosomes 2, 3, and 5 (referred to as PDX1.1, PDX1.2, and PDX1.3, respectively). Phylogenetic analysis of the PDX1 homologues in A. thaliana showed that PDX1.1 and PDX1.3 clustered with the homologues from the other dicots, whereas PDX1.2 was more divergent, and did not cluster with either the dicots or monocots. Expression analysis using quantitative PCR showed that PDX1.1 and PDX1.3 were highly expressed in A. thaliana rosettes, while PDX1.2 showed only low level expression. All three PDX1 genes and PDX2 were responsive to abiotic stressors including high light, chilling, drought, and ozone, however, the response of PDX1.2 was disparate from that of the other PDX genes, showing a lessened response to high light, chilling, and drought, but an increased response to ozone. Green fluorescent protein fusion studies demonstrated that PDX2 localizes in the nucleus and membranes of cells, consistent with recent published data for PDX1. Insight into regulation of the biosynthetic genes during abiotic stress could have important applications in the development of stress-tolerant crops.