To determine the role of the glycocalyx sialic acids residues in excitation-contraction coupling and the inotropic response to cardiotonic agents, we studied the effect of neuraminidase treatment on the response to ouabain, isoproterenol, calcium and reduced extracellular sodium in Langendorff preparations of adult guinea pig hearts. Neuraminidase treatment (0.01 unit/ml, 1 h) reduced the magnitude of the positive inotropic response to 2.5 X 10(-7) M ouabain and the maximum response to 5 X 10(-7) M ouabain by about 46% and 30%, respectively, but did not prevent ouabain toxicity. Neuraminidase treatment did not affect the contractility produced by calcium concentration alterations up to 5 mM calcium or the positive inotropic effect produced by lowering external sodium to as low as 80 mM. The inotropic response to as high as 10(-8) M isoproterenol was also not affected. The contractility response developed to calcium concentrations greater than 5 mM and to 5 X 10(-8) M isoproterenol were significantly reduced (P less than 0.05) by neuraminidase treatment. The content of sialic acids in neuraminidase-treated hearts used in the above concentration-response studies of ouabain, isoproterenol, calcium, and sodium was reduced by 70.7%, 66.1%, 65.6% and 66.2%, respectively. Neuraminidase treatment had no effect on basal (Na+ - K+)ATPase and Mg2+ - ATPase activities of (Na+ - K+)ATPase-containing membrane preparations of the guinea pig left ventricle. Neuraminidase treatment neither influenced the sensitivity of the enzyme (Na+ - K+)ATPase to ouabain inhibition nor did it affect the characteristics of [3H]ouabain binding to the preparation. These results suggest that the sialic acids of the glycocalyx in the guinea pig left ventricle play an important role in part of the inotropic response to subtoxic concentrations of ouabain.