Tubulin binding agents inhibit tubulin polymerization by actions at specific binding sites. CI-980 acts at the colchicine-binding site, which is distinct from the vinca-alkaloid binding site. We studied the actions of CI-980 in two models: neonatal rat myocytes in tissue culture and adult canine Purkinje fibers. In the first model, experiments on cell shortening and calcium signaling (using fluo3) showed that CI-980 increased the amplitude of both cell shortening and the Ca signal. The comparison drug, vinblastine, shared the effect on Ca signaling, but not that on cell shortening. In addition, high concentrations of CI-980 decreased the beating rate of spontaneously firing cell cultures. In canine Purkinje fibers, CI-980 decreased action potential amplitude (APA), Vmax, and conduction velocity and prolonged repolarization. It also decreased automaticity and suppressed delayed afterdepolarizations (DAD). These studies suggest that CI-980 is a novel compound in that it exerts antiarrhythmic effects on the AP but is positively inotropic. Whether all these actions derive from a primary effect on tubulin or whether they reflect action on both tubulin and transsarcolemmal ion channels remains to be determined.