The purH cDNA, encoding 5-aminoimidazole-4-carboxamide-ribonucleotide (AICAR) transformylase-inosine monophosphate cyclohydrolase (ATIC), was cloned by functional complementation of an Escherichia coli purH mutant using a chicken liver cDNA expression library. This represents the first report of the cloning of any eukaryotic ATIC-encoding cDNA (PurH). The avian ATIC mRNA is 2.3 kb long and encodes a protein with an Mr of 64,422. The deduced amino acid sequence is 36% identical to the bacterial purH-encoded enzymes from Bacillus subtilis and E. coli. The avian cDNA was expressed as a glutathione S-transferase (GST) fusion protein that was purified in a single step by affinity chromatography. A novel vector was employed which permits rapid and highly efficient cleavage of the GST fusion protein yielding 10 mg of purified PurH product per liter of bacterial culture. Km values were determined with the purified fusion protein utilizing AICAR and (6-R)N10-formyl-tetrahydrofolate as substrates. These values compare favorably with the isolated avian enzyme, supporting the idea that kinetic, as well as other physical properties of the recombinant fusion protein are similar to the native avian enzyme. Large quantities of purified enzyme and the ability to generate site-directed mutations should make mechanistic studies possible. The recombinant enzyme also affords a simple and reliable approach to identifying new antifolates.