An intra-arterially perfused working heart-brainstem preparation (WH-BP) was developed to allow studies into functionally identified cardiovascular and respiratory neurones in an in vitro milieu. This report provides that first description of this preparation. Evidence is presented indicating that the WH-BP: (1) spontaneously generates eupneic-like phrenic nerve activity, indicative of adequate oxygenation of the brainstem; (2) preserves the integrity of central coupling between the central respiratory rhythm generator and cardiac vagal motor neurones and, (3), allow an intracellular analysis of medullary cardio-respiratory neurones. The WH-BP may provide an advantaged environment for analysis of both synaptic and cellular mechanisms within the medulla that regulate cardio-respiratory activity.