Tryparedoxin has recently been discovered as a constituent of the trypanosomal peroxidase system catalysing the reduction of a peroxiredoxin-type peroxidase by trypanothione [Nogoceke et al. (1997) Biol. Chem. 378, 827-836] and has attracted interest as a potential molecular target for the development of trypanocidal agents. Here we describe the first isolation of a novel gene from Crithidia fasciculata encoding a different tryparedoxin designated tryparedoxin II. The deduced amino acid sequence of tryparedoxin II (accession number AF055986) differs substantially from the partial sequence reported for the tryparedoxin described previously and now renamed tryparedoxin I. It shares the sequence motif Vx3FSAxWCPPCR shown to represent the catalytic site in tryparedoxin I [Gommel et al. (1997) Eur. J. Biochem. 248, 913-918] with mouse nucleoredoxin (accession number X92750), and a thioredoxin-like gene product of Caenorhabditis elegans (accession number U23511). Depending on which ATG is considered functional as translation start codon, tryparedoxin II, with 150 or 165 amino acid residues, is 50% larger than the typical thioredoxins. The tryparedoxins appear phylogenetically related to the thioredoxins, but sequence similarities are restricted to the active site motifs and their intimate neighbourhood. His-tagged tryparedoxin II expressed in E. coli exhibited ping-pong kinetics in the trypanothione:peroxiredoxin assay with kinetic parameters (KM peroxiredoxin = 4.2 microM, KM trypanothione = 33 microM, Vmax/[E] = 952 min(-1)) similar to those reported for tryparedoxin I [Gommel et al. (1997) Eur. J. Biochem. 248, 913-918]. The co-existence of two distinct tryparedoxins in C. fasciculata suggests diversified biological roles of this novel type of protein, which in trypanosomatids may substitute for the pleiotropic redox catalyst thioredoxin.