Davis et al. proposed that the primary acoustic startle reflex is mediated by synapses in the ventral cochlear nucleus (VCN), lateral lemniscus (LL) and caudal pontine reticular formation (PnC). The collision test was used here to estimate the locations of synapses mediating startle-like responses in these sites, and the conduction times between and across these synapses. Conditioning (C) and test (T) pulses were delivered to pairs of sites in chloral hydrate anaesthetized rats, and current thresholds for, and latencies of, hindlimb EMG responses were determined. When sites are axonally connected, thresholds increase at the same positive and negative C-T intervals ('symmetric collision'), but when sites are connected by strong synapses, thresholds increase at asymmetric C-T intervals. (i) Asymmetric collisions between VCN and contralateral PnC centered on a C-T interval of + 0.5 ms suggest a monosynaptic connection in ventrolateral pons (VLP), near LL. (ii) Asymmetric collisions between VCN and contralateral medulla centered on a C-T interval of + 0.85 to + 1.0 ms suggest a disynaptic connection. (iii) Asymmetric collisions between VLP and ipsilateral medulla centered on a C-T interval of 0.2-0.4 ms suggest a monosynaptic connection in PnC, as shown previously in freely behaving animals [32]. (4) Symmetric collisions between VLP and rostral PnC, and between caudal PnC and medulla suggest fast axonal connections. Latency differences between electrode pairs were generally similar to collision-derived conduction times. From these, conduction times and transmission times were estimated for each axon bundle and synapse in the circuit.