A method which combined mechanical and enzymatic dissociation of brain parenchyma with Percoll density gradient centrifugation of the resulting tissue suspension was developed for the extraction of viable mononuclear inflammatory cells from the brains of mice. This method was used to extract mononuclear inflammatory cells from the brains of normal mice and mice chronically infected with the intracellular parasite Toxoplasma gondii. Infection with T. gondii was found to result in a 5- to 7-fold increase in the number of mononuclear cells which could be extracted from mouse brains. Immunocytochemical characterization of the extracted mononuclear cell fractions using monoclonal antibodies against T cell subsets and monocyte/macrophages revealed that the numbers of helper T cells, cytotoxic/suppressor T cells, and monocyte/macrophages present in mouse brain increased markedly after infection with T. gondii. This method may prove useful in identifying the cells responsible for inhibition of growth of tumor cells in the brain which has been observed after infection with T. gondii in mice.