Previously, we reported the characterization of pypAg-1, a novel protective membrane protein of Plasmodium yoelii-infected erythrocytes. Immunization studies indicated that pypAg-1 contained at least two protective epitopes. One of these determinants was associated with the N-terminal portion of pypAg-1, that also included a 220 amino acid domain unusually rich in tryptophan residues. Using sera from mice immunized against P. yoelii, we have identified a second related antigen, pypAg-3. The pypag-3 cDNA encodes a 43 kDa blood-stage protein that is also characterized by the presence of a 220 residue tryptophan-rich domain. Of particular interest, sequence comparisons revealed that 24 tryptophan residues are positionally conserved between pypAg-1 and pypAg-3. Otherwise, the two antigens share limited sequence similarity. Full-length recombinant pypAg-3 was expressed, purified and used to produce a high titer polyclonal rabbit antiserum. As with pypAg-1, immunofluorescence studies showed that pypAg-3 is expressed in the cytoplasm and associated with the membrane of P. yoelii infected erythrocytes. In addition, pypAg-1 and pypAg-3 appear to be secreted proteins, as both were detected in culture supernatants of P. yoelii-infected erythrocytes. Finally, metabolically labeled pypAg-1 and pypAg-3 secreted from parasitized cells bind to the surface of uninfected, normal mouse erythrocytes. As such, the conservation of the unusual tryptophan-rich domain between two blood-stage malarial proteins with similar biological properties suggests that it may be important for protein export and/or function.