Many factors influence neurodevelopment. However, their contribution to adult neural function is often unclear. This is often due to complex expression profiles, cell signalling, neuroanatomy, and a lack of effective tests to assess the function of neural circuits in vivo. Ephrin-A2 and ephrin-A5 are cell surface proteins implicated in multiple aspects of neurodevelopment. While the role of ephrin-As in visual, auditory and learning behaviours has been explored, little is known about their role in dopaminergic and neuromotor pathways, despite expression in associated brain regions. Here we probe the function of ephrin-A2 and ephrin-A5 in the development of the dopaminergic and neuromotor pathways using counts of tyrosine hydroxylase (TH) positive cells in the substantia nigra pars compacta (SNpc) and the ventral tegmental area (VTA), the acoustic startle reflex (ASR), and a measure of sensorimotor gating, prepulse inhibition (PPI). Analysis of the ASR and PPI in ephrin-A2 and/or ephrin-A5 knock-out mice revealed that both genes play distinct roles in mediating ASR circuits, but are unlikely to play a role in PPI. Knock-out of either gene resulted in robust changes in startle response magnitude and measures of startle onset and peak latencies. However, ephrin-A2 and ephrin-A5 regulate aspects of the ASR differently: ephrin-A2 KO mice have increased startle amplitude, increased sensitivity and reduced latency to startle, whilst ephrin-A5 KO mice show opposite effects. Neither of the gene knock outs affected PPI, despite ephrin-A5 KO mice showing changes in dopamine cell numbers in nuclei thought to regulate PPI. We propose that majority of the changes observed ephrin-A2 and ephrin-A5 KO mice appear to be mediated by the effects on motor neurons and their muscle targets, rather than changes in auditory sensitivity. © 2014 Elsevier B.V.