Migration of fluorescent DNA-labeled or 111Indium-labeled activated lymphocytes was studied in normal rat brain bearing surgically implanted cannulas. The migration of allogeneic cytotoxic T lymphocytes (CTL), derived from the DA rat (DA anti Fischer CTL), and of syngeneic concanavalin A (ConA)-activated lymphocytes (Fischer Con A blasts), was determined in Fischer rats between 2 h and 7 days post instillation into parietal brain. Whole body nuclear imaging indicated that the majority of the radiolabeled lymphocytes, either syngeneic or allogeneic, were present in the brain at 2 and 18 h. Autoradiography of brain slices demonstrated that label was located throughout the brain and in both hemispheres at all time points. By direct tissue radioassay, approximately 60% of the injected dose was present between 2 and 18 h; this decreased to 18% by day 7. By fluorescence microscopy, large numbers of lymphocytes were visible up to 3-4 days. The lymphocytes traveled from the instillation site into both cerebral hemispheres primarily following white matter tracts. Preferential localization of fluorescently labeled lymphocytes was seen in the corpus callosum, internal and external capsules, anterior commissures, lateral olfactory tracts, white matter connections in the caudate and putamen, mammillothalamic and optic tracts. Overall, gray matter contained fewer cells although perivascular spaces within it had high concentrations of cells, indicating these spaces may act as points of egress.