Abstract Introduction: Carbachol, an agonist at muscarinic receptors, exerts negative inotropic effects in human atrium. Carbachol can activate protein phosphatases (PP1 or PP2A). We hypothesized that cantharidin or sodium fluoride, inhibitors of PP1 and PP2A, might attenuate negative inotropic effects of carbachol. Methods: During bypass-surgery trabeculae carneae human atrial preparations (HAP) were obtained. These trabeculae were mounted in organ baths and electrically stimulated (1 Hz). Force of contraction was measured under isometric conditions. For comparison, we studied isolated electrically stimulated left atrial preparations (LA) from mice. Results: 100 µM cantharidin and 3 mM sodium fluoride increased force of contraction in LA (n = 5-8, p < 0.05) by 113 % ± 24.5 % and by 100 % ± 38.2 % and in HAP (n = 13-15, p < 0.05 ) by 625 % ± 169 % and by 196 % ± 23.5 %, respectively. Carbachol 1 µM alone exerted a rapid transient maximum negative inotropic in LA (n = 6) and HAP (n = 14) to 46.9 % ± 3.63 % and 19.4 % ± 3.74 %, respectively (p < 0.05). These negative inotropic effects were smaller in LA (n = 4-6) and HAP (n = 9-12) pretreated with 100 µM cantharidin and amounted to 58.0 % ± 2.27 % and 59.2 % ± 6.19 % or 3 mM sodium fluoride to 63.7 % ± 9.84 % and 46.3 % ± 5.69 %, (p<0.05). Conclusion: We suggest that carbachol, in part, exerts a negative inotropic effect in the human atrium by putatively stimulating the enzymatic activity of PP1 and/or PP2A.