Compounds that specifically inhibit S-adenosylhomocysteine hydrolase (SAHH; EC 3.3.1.1) interfere with the proliferation of Plasmodium malarial parasites, but efforts to identify the enzyme directly in parasite extracts have been unsuccessful. Here we report genetic and biochemical evidence for the presence of a gene encoding P. falciparum SAHH. The gene is transcribed as a 2.8-kilobase mRNA in erythrocytic stage parasites. Analysis of the open reading frame predicts a 53.9-kDa protein having conserved regions thought to be involved in NAD binding. The cDNA sequence has been incorporated into an Escherichia coli expression construct to confirm the function of the sahh product. Transformed E. coli cells produce a protein with a relative molecular weight of 56,000 which possesses SAHH activity as evidenced by the conversion of 3-deazaadenosine to S-3-deazaadenosylhomocysteine. Several amino acid residues that have been suggested to be at the SAHH active site in other organisms show nonconserved replacements in P. falciparum, suggesting that some current proposals for the enzyme mechanism may need to be revised. The structural differences between the P. falciparum and mammalian SAHH enzymes may foster innovative strategies for drug development against malaria.