1 The effects of antimuscarinic agents alone and in the presence of neostigmine on the contractile responses to exogenously applied cholinomimetics or (-)-noradrenaline were studied in the rat anococcygeus muscle.2 Atropine (1 x 10(-9) -1 x 10(-6)M) alone, in the presence of hexamethonium (1 x 10(-4)M), or phentolamine (1 x 10(-6)M), inhibited responses to acetylcholine but not to (-)-noradrenaline. The inhibitory effect with the higher concentrations of atropine (1 x 10(-8) - x 10(-6)M), was seen as an increase in the slopes of the concentration-response curves. Atropine (1 x 10(-8)M) alone inhibited the responses to methacholine and carbachol without altering the slopes of the concentration-response curves.3 Homatropine (1 x 10(-6)M) alone had no effect on responses to (-)-noradrenaline and inhibited responses to acetylcholine and methacholine. The inhibitory effect on responses to acetylcholine but not to methacholine, included an increase in the slopes of the concentration-response curves.4 Neostigmine (1 x 10(-6)M) alone had no effect on responses to (-)-noradrenaline and potentiated responses to acetylcholine and methacholine. The potentiating effect included an increase in the slopes of the concentration-response curves.5 In the presence of neostigmine (1 x 10(-6)M), atropine (1 x 10(-9)M - 1 x 10(-6)M) caused a parallel concentration-dependent shift of the concentration-response curves to acetylcholine. The pA(2) values, in the presence of neostigmine, were independent of the concentration of atropine and of the agonist (acetylcholine, methacholine, or carbachol) used. In the presence of neostigmine (1 x 10(-6)M), homatropine (1 x 10(-6)M) also failed to alter the slopes of the concentration-response curves to acetylcholine and was approximately 100 times less potent than atropine as an antimuscarinic agent.6 These results illustrate that, in the rat anococcygeus muscle, it is necessary to inhibit acetylcholinesterase before determining the relative potencies of antagonists at muscarinic receptors.