An IFAT was used to determine the prevalence of Neospora-specific IgG antibodies in serum from Alabama horses. Serum samples (n = 536) were from asymptomatic horses routinely submitted for equine infectious anaemia virus infection testing. We also subjected a 13-year-old horse with CNS disease to necropsy examination for isolation and in vitro cultivation of protozoal organisms. In antemortem tests, this horse was positive for antibodies to Neospora sp. in the IFAT and western immunoblot. Results of the prevalence survey indicated that IgG antibodies to Neospora were present in 62 (11.5%) of the 536 serum samples. Endpoint titres for the positive samples were 1:50 (35/6.5%), 1:100 (19/3.5%), 1:200 (7/1.3%) and 1:1600 (1/0.2%). Tachyzoites were first seen in cultured bovine turbinate cells 32 days after inoculation with spinal cord homogenates from the horse with CNS disease. Tachyzoites reacted with known N. caninum-positive serum from horses, cows, dogs and mice, but did not react with murine anti-Toxoplasma gondii or equine anti-Sarcocystis neurona serum. Ultrastructural features of tachyzoites and results of comparison of tachyzoite immunodominant proteins revealed that they were identical to those of N. hughesi, a species described recently from a naturally infected horse. The isolate recovered from the naturally infected horse in the present study (designated NA1) is thought to be an isolate of N. hughesi, although confirmation of this awaits additional molecular characterisation. These results provide some additional evidence that N. hughesi is a valid species and that Neospora infections in horses may occur in widely separated geographic regions of the United States.