Proline is one of the most common compatible osmolytes in water-stressed plants. The accumulation of proline in dehydrated plants is caused both by the activation of proline biosynthesis and by the inactivation of proline degradation; a decrease in the level of accumulated proline in rehydrated plants is caused both by the inhibition of proline biosynthesis and by the activation of proline degradation. The proline biosynthetic pathway has been well characterized, but the degradation of proline is poorly understood. Sequence analysis of an Arabidopsis cDNA clone, ERD5 (for early responsive to dehydration stress), isolated from plants dehydrated for 1 hr, revealed that it encodes a protein with identity to products of the yeast PUT1 (for proline utilization) gene (23.6% over 364 amino acids) and the Drosophila sluggish-A gene (34.5% over 255 amino acids). Their gene products are precursors of proline oxidases (dehydrogenase) (EC 1.5.99.8), which are the first enzymes involved in the conversion of proline to glutamic acid. Proline oxidase is localized in mitochondria. RNA gel blot analysis demonstrated that transcripts of the ERD5 gene were undetectable when plants had been dehydrated for 10 hr, but large amounts of the transcript accumulated when plants subsequently were rehydrated. Elevated levels of the transcript were also found in plants that had been incubated in a medium that contained proline. Immunologically, we showed that the product of ERD5 is localized in the mitochondrial fraction and accumulates in response to proline in cultured cells. Fusion genes for ERD5 and PUT1 complemented a put1 mutant of yeast, allowing put1 to grow with proline as the source of nitrogen. These results suggest that ERD5 encodes a precursor of proline dehydrogenase (oxidase), which is regulated at the level of mRNA accumulation in both dehydrated and rehydrated plants.