Inflammatory neurologic diseases are common in dogs, but establishing a definitive diagnosis often is difficult. Nucleated cell number and type in cerebrospinal fluid (CSF) rarely are suggestive of an etiologic agent. We speculated that CSF leukocyte immunophenotyping would be a useful adjunct in the investigation of canine inflammatory neurologic diseases by yielding more specific etiologic information. The goals of this study were to establish the feasibility of flow cytometric evaluation of individual canine CSF samples and to identify the cell distribution in healthy dogs. The mononuclear cell populations of paired blood and CSF samples from 23 healthy dogs were characterized by labeling of cells with antibodies against CD4, CD8alpha, CD21, and CD14 molecules and by flow cytometric analysis of their expression. The mean proportion of CD4+ and CD21+ cells was significantly higher in blood than in the CSF (P < .002 and P < .001, respectively). In contrast, the mean proportion of CD14+ and CD8a+ cells was not significantly different between blood and CSF (P = .5 and p = .9, respectively). These findings demonstrate differences in the distribution and function of mononuclear cells in the circulating venous and subarachnoid compartments in the dog.