Because of the similarity between human and canine narcolepsy, an animal model has emerged with which to investigate the neurochemical basis of the disease. In this study, in vivo differences in cerebral neurotransmitter function between normal and affected dogs were measured by comparing, before and after probenecid administration, the concentrations in cisternal cerebrospinal fluid of the monoamine metabolites 5-hydroxyindoleacetic acid, homovanillic acid, 3,4-dihydroxyphenylacetic acid, and 3-methoxy-4-hydroxyphenylethylene glycol. Cisternal cerebrospinal fluid was collected from the cisterna magna of anaesthetized animals, and samples were analyzed for the metabolites and probenecid using combined gas chromatography-mass spectrometry. The concentrations of both the conjugated and non-conjugated forms of each of the metabolites were determined in all samples and the role of conjugation in cerebral monoamine metabolism is discussed. Before probenecid treatment, comparisons of metabolite concentrations in samples from normal versus narcoleptic dogs showed significantly lower amounts of the dopamine and of the 5-hydroxytryptamine metabolites in CSF from the affected animals. After probenecid administration, the concentrations in the CSF of all the metabolites increased. However, the amounts of 5-hydroxyindoleacetic acid, and the norepinephrine metabolite 3-methoxy-4-hydroxyphenylethylene glycol, in samples from narcoleptic dogs were significantly lower than those found in samples from normal dogs. The implications of these results for our understanding of the neurochemical basis of REM sleep initiation and for treatment of narcolepsy/cataplexy are discussed.