ATP-sulfurylase, the first enzyme of sulfate assimilation, catalyzes the formation of adenosine-5'-phosphosulfate from ATP and sulfate. Here we report that the higher plant, Arabidopsis thaliana, contains a three-member, expressed gene family encoding plastid localized forms of ATP sulfurylase. Three cDNAs from A. thaliana, designated APS1, APS2, and APS3, were isolated by their ability to functionally complement a met3 (ATP sulfurylase) mutant strain of Saccharomyces cerevisiae (yeast). The nucleotide sequence of APS1 was reported previously (1). APS2 and APS3, reported here, have 476- and 465-codon open-reading frames encoding 53.6- and 52.0-kDa polypeptides, respectively. The translation products of both clones are highly homologous to APS1 (66 and 86% identity, respectively) over their entire lengths, including amino terminal sequences resembling transit peptides for plastid localization. Both clones are less homologous to MET3 (25 and 30% identity, respectively). Genomic blot analysis of A. thaliana revealed only three genes with homology to the APS cDNAs and RNA blot analysis showed that APS1 is the most highly expressed member of this gene family. The APS polypeptides share homology with ATP-sulfurylases from fungi, a marine worm and a chemoautotrophic bacterium, but, not from Escherichia coli or Rhizobium meliloti. Analysis of recombinant APS3 showed that the protein is structurally and kinetically similar to fungal ATP-sulfurylase, but very different from the E. coli enzyme. The APS3 polypeptide is a homotetramer with specific activities (mumol primary product x mg protein-1 at pH 8.0, 25 degrees C) for 2.9 for APS synthesis, 30.1 for molybdolysis, and 48.7 for ATP synthesis. Despite the sequence, structural, and kinetic differences between higher plant and E. coli ATP-sulfurylases, APS2 and APS3 are able to functionally complement E. coli cysD and cysN (ATP-sulfurylase) mutant strains.