We were successful in developing cortex-striatum-mesencephalon organotypic cultures from the rat brain after 4–9 weeks in vitro. A modification of the ‘roller tube’ technique was employed where slices were embedded in a plasma/thrombin clot onto a Millicell membrane on a coverslip. The underlying membrane provided high mechanical stability during culturing, which reduced the likelihood of deterioration of the cultures. Tyrosine hydroxylase immunoreactivity was used to label dopamine neurons and axonal innervation into the cortical and striatal culture. The electrophysiological responses of striatal medium-sized spiny neurons to cortical, striatal and mesencephalic stimulation were characterized.