During January 2007, blood samples were collected from 552 healthy horses from nine different regions of the Czech Republic. Sera were tested for serum antibodies to Neospora caninum by a competitive-inhibition enzyme-linked immunosorbent assay and confirmed by an indirect fluorescent antibody test. The same samples were tested for serum antibodies against Toxoplasma gondii by a latex agglutination test. In total, 131 of 552 (24%) horses reacted positively for Neospora antibodies in competitive-inhibition enzyme-linked immunosorbent assay; seven of them had > or =50% of inhibition. Samples were confirmed in indirect fluorescence test, and only two samples were positive with final titres 50 and 100, while others were negative. Antibodies against T. gondii were found in 125 (23%) horses. This is the first serologic survey for Neospora spp. antibodies performed on horses in the Czech Republic.