The serotonin metabolite, 5-hydroxyindoleacetic acid (5-HIAA) and the dopamine metabolite, homovanillic acid (HVA) in the cerebrospinal fluid (CSF) of seven clinically normal horses were evaluated with reverse phase high pressure liquid chromatography and electrochemical detection. Comparisons of the neurotransmitter metabolite concentrations were made on CSF collected simultaneously from the atlanto-occipital and lumbosacral regions. There were significantly higher amounts of 5-HIAA and HVA in atlanto-occipital CSF than in lumbosacral CSF. Mean 5-HIAA concentrations in atlanto-occipital and lumbosacral CSF were 206.1±37.0 nmol/L and 68.5±12.6 nmol/L, respectively. The mean HVA concentration in atlanto-occipital CSF was 254.4±50.4 nmol/L. No HVA was detected in lumbosacral CSF. In contrast, significantly greater protein concentrations were present in lumbosacral CSF than in atlanto-occipital CSF. Lumbosacral and atlanto-occipital CSF contained 32.1±3.7 mg protein/dl and 25.0±1.3 mg protein/dl, respectively. Thus, between the atlanto-occipital and lumbosacral space of the horses, a rostrocaudal gradient existed for neurotransmitter metabolites and a caudorostral gradient existed for protein.