A cDNA library derived from mRNA of tachyzoites of Neospora caninum (NC-Liverpool strain) was screened with antisera from a cow naturally infected with N. caninum. The DNA sequence of 1 recombinant isolated predicted a significant protein sequence homology of the gene product to the 28 kDa (GRA2) antigen of Toxoplasma gondii. Studies on the N. caninum gene coding for this antigen demonstrated the presence of a single intron flanked by 2 exons; the gene was also highly expressed in culture-derived tachyzoites. The antigen was expressed in Escherichia coli; when injected into mice it stimulated the production of antibodies which detected a 29 kDa antigen of N. caninum. Secondary structure predictions made for the N. caninum protein showed support for several amphipathic helices separated by loops and turns. The available evidence indicates maintenance of protein secondary structure, and not DNA or amino acid sequence, has occurred during the evolution of GRA2 proteins in N. caninum and T. gondii.