Transforming growth factors-β1 (TGF-β1), -2, and -3 form a small group of related proteins involved in the regulation of proliferation, differentiation, and survival of various cell types. Recently, TGF-βs were also demonstrated to be neuroprotective. In the present study, we investigated their distribution in the rat brain as well as their expression following middle cerebral artery occlusion. Probes were produced for all types of TGF-βs, and in situ hybridization was performed. We demonstrated high TGF-β1 expression in cerebral cortex, hippocampus, central amygdaloid nucleus, medial preoptic area, hypothalamic paraventricular nucleus, substantia nigra, brainstem reticular formation and motoneurons, and area postrema. In contrast, TGF-β2 was abundantly expressed in deep cortical layers, dentate gyrus, midline thalamic nuclei, posterior hypothalamic area and mamillary body, superior olive, areas of monoaminergic neurons, spinal trigeminal nucleus, dorsal vagal complex, cerebellum, and choroid plexus, and a high level of TGF-β3 mRNA was found in cerebral cortex, hippocampus, basal amygdaloid nuclei, lateral septal nucleus, several thalamic nuclei, arcuate and supramamillary nuclei, superior colliculus, superior olive, brainstem reticular formation and motoneurons, area postrema, and inferior olive. Focal brain ischemia induced TGF-βs with markedly different expression patterns. TGF-β1 was induced in the penumbral region of cortex and striatum, whereas TGF-β2 and -β3 were induced in different layers of the ipsilateral cortex. The expression of the subtypes of TGF-βs in different brain regions suggests that they are involved in the regulation of different neurons and bind to different latent TGF-β binding proteins. Furthermore, they might have subtype-specific functions following ischemic attack. J. Comp. Neurol. 518:3752–3770, 2010. © 2010 Wiley-Liss, Inc.