We have established the feasibility of using Neospora caninum as a heterologous system for the expression of genes from the closely-related parasite Toxoplasma gondii. Plasmid constructs containing the lacZ gene from Escherichia coli driven by T. gondii promoters were electroporated into N. caninum parasites, and expression of β-galactosidase (β-Gal) activity was assayed enzymatically. In transient assays, expression of β-Gal driven by the GRA1 promoter was approximately 10-fold higher than the expression obtained with the SAG1 promoter. Enzyme activity was not detected when N. caninum parasites were transfected with a promoterless lacZ construct. Transfection of N. caninum with complete genomic clones of SAG1 or GRA2 from T. gondii yielded parasites that transiently expressed the respective gene products, as detected by immunofluorescence and Western blot. Additionally, these transiently expressed T. gondii proteins appeared by immunofluorescence localization and Triton X-114 partitioning to be correctly targeted in both extracellular and intracellular N. caninum parasites. Heterologous gene expression should be useful for studying the function of specific gene products and may facilitate the identification of genes responsible for the phenotypic differences observed between these two closely-related apicomplexan parasites.