AbstractWe studied the presynaptic muscarinic autoreceptor subtypes controlling ACh release and their relationship with voltage‐dependent calcium channels in the neuromuscular synapses of theLevator auris longusmuscle from adult (30–40 days) and newborn (3–6 and 15 days postnatal) rats. Using intracellular recording, we studied how several muscarinic antagonists affected the evoked endplate potentials. In some experiments we previously incubated the muscle with calcium channel blockers (nitrendipine, ω‐conotoxin‐GVIA and ω‐Agatoxin‐IVA) before determining the muscarinic response. In the adult, the M1 receptor‐selective antagonist pirenzepine (10 µm) reduced evoked neurotransmission (≈ 47%). The M2 receptor‐selective antagonist methoctramine (1 µm) increased the evoked release (≈ 67%). Both M1‐ and M2‐mediated mechanisms depend on calcium influx via P/Q‐type synaptic channels. We found nothing to indicate the presence of M3 (4‐DAMP‐sensitive) or M4 (tropicamide‐sensitive) receptors in the muscles of adult or newborn rats. In the 3–6‐day newborn rats, pirenzepine reduced the evoked release (≈ 30%) by a mechanism independent of L‐, N‐ and P/Q‐type calcium channels, and the M2 antagonist methoctramine (1 µm) unexpectedly decreased the evoked release (≈ 40%). This methoctramine effect was a P/Q‐type calcium‐channel‐dependent mechanism. However, upon maturation in the first two postnatal weeks, the M2 pathway shifted to perform the calcium‐dependent release‐inhibitory activity found in the adult. We show that the way in which M1 and M2 muscarinic receptors modulate neurotransmission can differ between the developing and adult rat neuromuscular synapse.