Neuronal acetylcholine nicotinic receptors (nAChR) are composed of 12 subunits (α2‐10, β2‐4), of which α3, α5, α7, β2 and β4 subunits are known to exist in the autonomic nervous system (ANS). α5 subunits possess unique biophysical and pharmacological properties. The present study was undertaken to examine the functional role and pharmacological properties of the nAChR α5 subunits in the ANS using mice lacking α5 nAChR subunits (α5‐/‐). These mice grew to normal size showing no obvious physical or neurological deficit. They also showed normality in thermoregulation, pupil size and resting heart rate under physiological conditions. The heart rate and rectal temperature did not differ between α5‐/‐ and wild‐type mice during exposure to cold stress. An impairment of cardiac parasympathetic ganglionic transmission was observed during high frequency vagal stimulation, which caused cardiac arrest in all wild‐type animals while α5‐/‐ mice were more resistant. Deficiency of α5 subunits strikingly increased the sensitivity to a low concentration of hexamethonium, leading to a nearly complete blockade of bradycardia in response to vagal stimulation. Such a concentration of hexamethonium only slightly depressed the effects of vagal stimulation in control mice. Deficiency of α5 subunits significantly increased ileal contractile responses to cytisine and epibatidine. These results suggest that α5 subunits may affect the affinity and sensitivity of agonists and antagonists in the native receptors. Previous studies revealed that α5 subunits form functional receptors only in combination with other α and β subunits. Thus, the data presented here imply that α5 subunits modulate the activity of nAChR in autonomic ganglia in vivo.