1. To examine the functional importance of the pre-Bötzinger complex for breathing we micro-injected, under in vivo conditions, the calcium channel blocker omega-conotoxin GVIA and the sodium channel blocker tetrodotoxin (TTX) into the ventrolateral medulla of adult cats, while monitoring respiratory rhythmic motor output in the phrenic nerve. 2. omega-Conotoxin GVIA caused a highly localized synaptic ablation by blocking presynaptic N-type calcium channels. When injecting 5-60 fmol omega-conotoxin GVIA unilaterally, the amplitude of phrenic nerve activity decreased bilaterally and sometimes disappeared, indicating central apnoea. These effects were reversible and could only be induced in a very localized area of the pre-Botzinger complex. By injecting omega-conotoxin GVIA several times during an experiment and analysing the areas where injections affected respiratory activity, it was possible to map exactly the anatomical extent of the area critical for respiratory rhythm generation. 3. Following the precise localization of the pre-Bötzinger complex with omega-conotoxin GVIA, we injected TTX to induce an irreversible inactivation of this region. TTX injected unilaterally into the pre-Bötzinger complex irreversibly reduced the amplitude of phrenic nerve activity. Bilateral TTX injections eliminated respiratory rhythmic activity, causing a persistent central apnoea. 4. After bilateral lesioning of the pre-Bötzinger complex, it was still possible to induce gasping during hypoxia or asphyxia, indicating that respiration and gasping are generated by two different neuronal networks. 5. We propose that omega-conotoxin GVIA as employed in this study to investigate the functional role of the pre-Bötzinger complex can also be used as a general pharmacological approach to map other neuronal networks. We call this the 'omega-conotoxin GVIA tracing' method.