This study was designed to determine the relative levels of gene transcription of selected pathogens and cytokines in the brain and spinal cord of 12 horses with equine protozoal myeloencephalitis (epm), 11 with equine herpesvirus type 1 (ehv-1) myeloencephalopathy, and 12 healthy control horses by applying a real time pcr to the formalin-fixed and paraffin-embedded tissues. Total rna was extracted from each tissue, transcribed to complementary dna (cdna) and assayed for Sarcocystis neurona, Neospora hughesi, ehv-1, equine gapdh (housekeeping gene), tumour necrosis factor (tnf)-α, interferon (ifn)-γ, interleukin (il)-1β, il-2, il-4, il-6, il-8, il-10 and il-12 p40. S neurona cdna was detected in the neural tissue from all 12 horses with epm, and two of them also had amplifiable cdna of N hughesi. The relative levels of transcription of protozoal cdna ranged from 1 to 461 times baseline (mean 123). All the horses with ehv-1 myeloencephalopathy had positive viral signals by pcr with relative levels of transcription ranging from 1 to 1618 times baseline (mean 275). All the control horses tested negative for S neurona, N hughesi and ehv-1 cdna. The cytokine profiles of each disease indicated a balance between pro- and anti-inflammatory markers. In the horses with epm the pro-inflammatory Th1 cytokines (il-8, tnf-α and ifn-γ) were commonly expressed but the anti-inflammatory Th2 cytokines (il-4, il-6 and il-10) were absent or rare. In the horses with ehv-1 the proinflammatory cytokine il-8 was commonly expressed, but il-10 and ifn-γ were not, and tnf-α was rare. Tissue from the control horses expressed only the gene gapdh.