Three different cDNAs, Prh-19, Prh-26, and Prh-43 [3-phosphoadenosine-5-phosphosulfate (PAPS) reductase homolog], have been isolated by complementation of an Escherichia coli cysH mutant, defective in PAPS reductase activity, to prototrophy with an Arabidopsis thaliana cDNA library in the expression vector YES. Sequence analysis of the cDNAs revealed continuous open reading frames encoding polypeptides of 465, 458, and 453 amino acids, with calculated molecular masses of 51.3, 50.5, and 50.4 kDa, respectively, that have strong homology with fungal, yeast, and bacterial PAPS reductases. However, unlike microbial PAPS reductases, each PRH protein has an N-terminal extension, characteristic of a plastid transit peptide, and a C-terminal extension that has amino acid and deduced three-dimensional homology to thioredoxin proteins. Adenosine 5-phosphosulfate (APS) was shown to be a much more efficient substrate than PAPS when the activity of the PRH proteins was tested by their ability to convert 35 S-labeled substrate to acid-volatile 35 S-sulfite. We speculate that the thioredoxin-like domain is involved in catalytic function, and that the PRH proteins may function as novel ''APS reductase'' enzymes. Southern hybridization analysis showed the presence of a small multigene family in the Arabidopsis genome. RNA blot hybridization with gene-specific probes revealed for each gene the presence of a transcript of Ϸ1.85 kb in leaves, stems, and roots that increased on sulfate starvation. To our knowledge, this is the first report of the cloning and characterization of plant genes that encode proteins with APS reductase activity and supports the suggestion that APS can be utilized directly, without activation to PAPS, as an intermediary substrate in reductive sulfate assimilation.Sulfur in its reduced form plays an important role in plant metabolism being involved in the biosynthesis of a wide range of primary and secondary S-containing metabolites. However the pathway of reductive assimilation of inorganic sulfate to sulfide in higher plants is the subject of some controversy (reviewed in ref. 1). After sulfate uptake, and its activation to adenosine 5Ј-phosphosulfate (APS) (5Ј-adenylylsulfate) by the enzyme ATP sulfurylase, it has been unclear whether APS is further metabolized by the enzyme APS sulfotransferase to ''bound-sulfite'' and then reduced to ''bound sulfide'' by a thiosulfonate reductase or whether the ''free-sulfite'' pathway, found in most enterobacteria and certain fungi and yeast, operates. In this latter pathway APS is converted to 3Ј-phosphoadenosine-5Ј-phosphosulfate (PAPS) (3Ј-phosphoadenylylsulfate) by APS kinase and PAPS is reduced to free sulfite by PAPS reductase. Sulfite is then reduced to sulfide by sulfite reductase. Discovery of a thioredoxin-dependent PAPS reductase activity in spinach (2) and the more recent suggestion that APS sulfotransferase activity is a side-reaction of APS kinase (3) provide strong support for the notion that the ''free-intermediate'' pathway operates not only in...