The acoustic startle response is an important mammalian model for studying the cellular mechanisms of emotions and learning. Lesions in the superior olivary complex have been shown to attenuate the amplitude of the acoustic startle response, thus a substantial contribution of these neurons to the startle response was proposed. We here explored the putative connection of olivary neurons to the startle-mediating giant neurons in the reticular formation in rat brain slices in vitro. Tracing studies and electrical stimulation in the superior olivary complex revealed a strong connection; however it was not possible to distinguish between olivary neurons and traversing auditory fibres. Glutamate uncaging in the olivary complex excited a subpopulation of olivary neurons but never PnC giant neurons, as shown by patch-clamp recordings. This clearly contradicts an excitatory connection from olivary neurons to PnC giant neurons and thus an involvement of the superior olivary complex in eliciting a startle response.