The expression of c-fos protein was examined by immunohistochemistry in serial sections of brainstem following the instillation of either autologous arterial blood (0.3 ml) or mock cerebrospinal fluid (0.3 ml) through a catheter placed in the cisterna magna, or following catheter placement alone in pentobarbital-anesthetized Sprague-Dawley rats. After injection, blood was distributed within the subarachnoid space surrounding the brainstem and in the region of the circle of Willis. c-fos protein-like immunoreactivity was present at 1 h, peaked at 2 h and decreased by 8 h. At 2 h, immunoreactivity was strongly expressed within trigeminal nucleus caudalis (lamina I, IIo), as well as within nucleus of the solitary tract, area postrema, ependyma, pia mater and arachnoid in every animal. Moderate labeling was found in parabrachial nucleus, medullary lateral reticular nucleus and central gray. Sparse labeling was present in trigeminal nucleus caudalis (lamina III-V) and trigeminal nucleus interpolaris; few or no labeled cells were detected in other parts of the trigeminal nuclear complex, thalamus, cerebral cortex, cerebellar cortex or trigeminal ganglion. The number of positive cells was not related to the volume of injectate but was related to the amount of injected blood. The density of cell labeling evoked by injecting mock cerebrospinal fluid or after catheter placement was markedly lower than after blood in all brainstem areas. The number of labeled cells was greatly reduced within trigeminal nuclear complex, parabrachial nucleus and medullary lateral reticular nucleus, but not within the nucleus of the solitary tract, area postrema or ependyma when blood was injected into adult animals in which unmyelinated C-fibers were destroyed by neonatal capsaicin treatment. Similar results were obtained after blood was instilled into the cisterna magna of rats in which meningeal afferents were chronically sectioned at the ethmoidal foramen bilaterally. We conclude that blood in the subarachnoid space is an effective stimulus for activating c-fos expression within subpopulations of brainstem neurons. Activation within trigeminal nucleus caudalis is mediated in large part by excitation of small-caliber meningeal afferents (trigeminovascular fibers), whereas c-fos expression within nucleus of the solitary tract and area postrema may reflect direct stimulation of blood or blood products, or possibly the response to autonomic activation from noxious stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)