The purpose of this study was to determine the effect of blood contamination of cerebrospinal fluid (CSF) on the results of indirect fluorescent antibody tests (IFATs) for Sarcocystis neurona and Neospora hughesi. The in vitro study used antibody-negative CSF collected from non-neurologic horses immediately after euthanasia and blood samples from 40 healthy horses that had a range of IFAT antibody titers against S. neurona and N. hughesi. Serial dilutions of whole blood were made in seronegative CSF to generate blood-contaminated CSF with red blood cell (RBC) concentrations ranging from 10 to 100,000 RBCs/microl. The blood-contaminated CSF samples were then tested for antibodies against both pathogens using IFAT. Blood contamination of CSF had no detectable effect on IFAT results for S. neurona or N. hughesi at any serologic titer when the RBC concentration in CSF was <10,000 RBCs/microl. At concentrations of 10,000-100,000 RBCs/microl of CSF, positive CSF results (IFAT titer >or=5) for S. neurona and N. hughesi were detected only when the corresponding serum titers were >or=160 and >or=80, respectively. The IFAT performed on CSF is reliable for testing horses for equine protozoal myeloencephalitis caused by S. neurona or N. hughesi, even when blood contamination causes the RBC concentration in CSF to be up to 10,000 RBCs/microl.