1. This report provides the first description of respiratory network activity within the ventrolateral medulla of the mature mouse obtained from a unique working heart-brainstem preparation (WHBP). 2. In the WHBP three distinct respiratory phases were evident in recordings of both phrenic and vagal efferent nerves. These included a ramp inspiratory (I) discharge, post-inspiratory (PI) activity and a silent or expiratory interval (E2). 3. Extracellular recordings were made from different types of respiratory neurones located within, or in close proximity to, the nucleus ambiguus. Based on firing patterns and phase relative to phrenic nerve discharge, respiratory neurone types, including pre-inspiratory (PreI), early-inspiratory, throughout inspiratory (I), late-inspiratory, post-inspiratory (PI) and stage II expiratory or E2 neurones were characterized. 4. Intracellular recordings were made from four types of respiratory neurones (PreI, I, PI and E2 neurones). PreI neurones were depolarized maximally during the E2-inspiratory transition. I neurones exhibited a ramp depolarization which started either before or at the onset of phrenic discharge. Based on the kinetics of the inspiratory-related hyperpolarizations and duration of discharge, two types of PI neurones were found (rapidly adapting and slowly adapting). E2 neurones were hyperpolarized during both the inspiratory and post-inspiratory phases. 5. Phase-dependent chloride-mediated inhibition was studied in PreI, PI and E2 neurones and included: late inspiratory inhibition of PreI neurones; inspiratory-related inhibition of PI and E2 neurones; and post-inspiratory inhibition of PreI and E2 neurones. In addition, pre-inspiratory inhibition of PI neurones was also demonstrated. 6. The WHBP appears to be viable for analysing reflex, synaptic and cellular mechanisms regulating respiratory activity in an in vitro milieu. The synaptic organization of the respiratory network of the mouse appears comparable to that of the rat and cat. The possibility of a mutual inhibitory interaction between PreI and PI neurones is discussed in terms of the functional organization of the respiratory network in the mouse.