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Ascorbate (Asc) is an essential molecule in many aspects of development and stress responses in plants and animals. Cytochromes b561 (cyts b561) are tightly coupled to Asc homeostasis. These proteins are found in mammalian tissues, where they are involved in the regeneration of Asc, serving the synthesis of catecholamine neurotransmitters, and in intestinal iron reduction. Plant genomes encode homologous membrane-associated, Asc-reducible cyts b561. The expression of these proteins in plants, however, has so far not been studied. We have now examined the expression of two Arabidopsis thaliana cyt b561-encoding genes—Artb561-1 and Artb561-2—using relative-quantitative RT-PCR and in situ hybridization (ISH) techniques. The genes show overlapping and distinct tissue- and organ-specific expression patterns. Transcripts of both genes are found in leaf epidermal cells, and expression seems to correlate with leaf maturation and cessation of cell elongation. Both genes are also expressed in the epidermal cell layer of stems and roots in the L1 layer of the shoot apex, in the vascular system of leaves, stems and roots, and in the root pericycle. In addition, Artb561-1 is expressed in the root cap, whereas Artb561-2 mRNA is found in the epidermis of lateral roots, in the root meristem, and in unfertilized ovules. These observations provide important information for the elucidation of the physiological function of cyts b561 in plants.