Isoproterenol (5×10−6 m ) was perfused into a cat heart Langendorff preparation until a maximum positive inotropic response was obtained. Ouabain (9.6×10−7 m ), increased frequency of stimulation, an ionophore RO2-2985 (X537A) and “high” calcium were used to obtain similar increases in force of contraction. TnI was rapidly purified and the phosphate to protein ratios were determined. While isoproterenol, ouabain, calcium, RO2-2985 (RO) and increased frequency produced positive inotropy, only isoproterenol and RO effected an increase in protein-bound phosphate associated with TnI. It was concluded that isoproterenol and RO, but not other perturbations, stimulate phosphorylation of TnI. It is clear from the data, therefore, that phosphorylation of TnI may not be an obligatory requirement for positive inotropy.