The complete nucleotide (nt) sequence of the PEPCK gene encoding Trypanosoma cruzi phospho-enol-pyruvate carboxykinase (PEPCK; ATP dependent, EC 220.127.116.11) has been determined. The predicted primary sequence has 473 amino acids (aa) with a calculated molecular mass of 52.5 kDa. The ubiquitous spliced leader is present at nt position -60 from the AUG start codon in PEPCK mRNA; the coding region is followed by a long 3'-non-coding region of 777 nt. Northern and Southern blot analysis showed that the PEPCK mRNA is 2.7 kb long and that the PEPCK gene is polymorphic in T. cruzi, with more than one copy in the genome of the epimastigote form. Comparison of the available aa sequences of ATP(protozoa, yeast and bacteria)- and GTP(vertebrates, insects, helminths and fungi)-dependent PEPCKs showed that the former lack two characteristic, highly conserved regions present in the GTP-dependent enzymes: one is associated with the binding of PEP while the second is frequently labeled as 'catalytic' and contains a conserved Cys residue of unusual reactivity. On the other hand, two consensus sequences with conserved predicted secondary structure were identified in all PEPCKs, independent of their nt specificity; one of them is a divalent metal-binding site previously identified in pyruvate kinase by X-ray crystallographic studies.