To assess when vestibulosympathetic projections become functional postnatally, and to establish a preparation in which vestibulosympathetic circuitry can be characterized more precisely, we used an optical approach to record VIIIth nerve-evoked synaptic inputs to thoracic sympathetic preganglionic neurons (SPNs) in newborn mice. Stimulation of the VIIIth nerve was performed in an isolated brainstem-spinal cord preparation after retrogradely labelling with the fluorescent calcium indicator Calcium Green 1-conjugated dextran amine, the SPNs and the somatic motoneurons (MNs) in the thoracic (T) segments T2, 4, 6, 8, 10 and 12. Synaptically mediated calcium responses could be visualized and recorded in individual SPNs and MNs, and analysed with respect to latency, temporal pattern, magnitude and synaptic pharmacology. VIIIth nerve stimulation evoked responses in all SPNs and MNs investigated. The SPN responses had onset latencies from 90 to 200 ms, compared with much shorter latencies in MNs, and were completely abolished by mephenesin, a drug that preferentially reduces polysynaptic over monosynaptic transmission. Bicuculline and picrotoxin, but not strychnine, increased the magnitudes of the SPN responses without changing the onset latencies, suggesting a convergence of concomitant excitatory and inhibitory synaptic inputs. Lesions strategically placed to test the involvement of direct vestibulospinal pathways versus indirect pathways within the brainstem showed that vestibulosympathetic inputs in the neonate are mediated predominantly, if not exclusively, by the latter. Thus, already at birth, synaptic connections in the vestibulosympathetic reflex are functional and require the involvement of the ventrolateral medulla as in adult mammals.