Ten dogs were subjected to total extrinsic cardiac denervation. The negative inotropic responses of their hearts were compared with those of nine normal controls with respect to acetylcholine (ACh; 0.1-1.0 micrograms) and nicotine (0.5-100 micrograms) administered intra-coronary. The dogs were on cardiopulmonary bypass and atrial and ventricular contractility were measured by means of a four-chamber isovolumic technique. At the end of this phase of the experiment, atrial strips were removed for in vitro evaluation of negative inotropic responses to Ach (1 X 10(-8) to 1 X 10(-6) M) and nicotine (1 X 10(-6) to 1 X 10(-4) M). Both in vivo and in vitro methods demonstrated a leftward shift of the dose-response curve for nicotine in denervated hearts, indicative of denervation hypersensitivity. Hypersensitivity to ACh was seen in atria in vitro but not in vivo. An additional 13 dogs were used only for in vitro studies. There was no difference in response between these dogs and those which had been used for both in vivo and in vitro studies. The results support the in vivo observation that the intrinsic cardiac neurons become supersensitive to nicotinic activation after extrinsic denervation. In addition, in vitro testing revealed atrial hypersensitivity to ACh which was not detected by in vivo methods. Inasmuch as the in vivo and the in vitro testing were done on the same animals, a method for determining equivalence for in vivo doses and in vitro concentrations is presented.