A previously unknown interaction between acetylcholine (ACh) and adrenergic mechanisms is described, which increases the likelihood of a physiological association between the two divisions of the autonomic nervous system. In particular, ACh had a novel effect to disengage the neuronal mechanism that is purported to regulate the release of norepinephrine from sympathetic nerves. Brief exposure to ACh (1.4 X 10(-7) M-1.4 X 10(-5) M) inhibited the stimulation-evoked release of [3H]norepinephrine from guinea pig atria and ureter and rabbit aorta but higher concentrations (8.8 X 10(-5) or 1.4 X 10(-4) M) or prolonged exposure to moderate concentrations either had no visible effect or enhanced release. ACh blocked the ability of yohimbine, the presynaptic alpha receptor antagonist, to enhance the liberation of 3H-transmitter during field stimulation at 2 and 5 Hz, and it did so in all three of the test tissues. This effect was not attributable to a direct competition between ACh and yohimbine for presynaptic alpha sites and ACh did not act like yohimbine to increase transmitter release. The antagonistic effect of ACh bore no relation to the direct effect of ACh on adrenergic neurotransmitter release and occurred regardless of whether ACh itself inhibited, enhanced or did not affect transmitter liberation. Atropine blocked the effect of ACh on 3H-transmitter efflux and restored the capacity of yohimbine to enhance transmitter release. Inhibition of neurotransmitter release by norepinephrine was partially antagonized by ACh and this antagonism was also countered by atropine. Enhancement of norepinephrine release by phenoxybenzamine was also blunted by ACh. These findings cannot be incorporated into a model of neurotransmitter regulation that interprets the enhancement of norepinephrine release by adrenergic antagonists as the result of interruption of an on-going negative feedback system. The action of yohimbine appears linked to activation of presynaptic sites and not simply to their passive occupancy. A working model is offered to account for the interaction between ACh and adrenergic antagonists.