Currently, most undergraduate textbooks that cover the autonomic nervous system retain the concept that autonomic nerves release either acetylcholine or norepinephrine. However, in recent years, a large volume of research has superseded this concept with one in which autonomic nerves normally release at least one cotransmitter along with a dominant transmitter that may or may not be acetylcholine or norepinephrine. Cotransmission involving the simultaneous release of norepinephrine, ATP, and neuropeptide Y can easily be demonstrated in an isometric ring preparation of the rat tail artery, which is described here. The experiment clearly demonstrates the principle of cotransmission but allows more advanced concepts in autonomic cotransmission to be addressed.