Neospora hughesi n. sp. was isolated from the central nervous system tissue of an adult equine (Equus caballus) from California. The tachyzoites are crescent-shaped, approximately 2 x 5 microm (1.8-3.0 x 4.0-7.0 microm), with characteristic apical complex structures consisting of an anterior polar ring, conoid, numerous rhoptries filled with a uniform electron-dense material, and 22 microtubules extending posteriorly from the polar ring. Comparison of N. hughesi to canine and bovine Neospora caninum isolates showed phenotypic differences in immunoreactive proteins. Molecular analysis of the small subunit ribosomal RNA gene revealed no differences in the nucleotide sequence between N. hughesi and N. caninum isolates examined. However, the internal transcribed spacer I region revealed 7 nucleotide base differences between N. hughesi and N. caninum isolates (CN1 and BPA1) analyzed in this study. The existence of nucleotide base differences in the internal transcribed spacer regions suggests that this region may be a genetic marker for discriminating species within the genus Neospora. The ultrastructural, antigenic, and molecular data support distinction of N. hughesi as a new species, separate from N. caninum, the only recognized species in this genus.