Ventricular myocytes express Gαq-coupled receptors that can mediate enhanced contractility by increasing the sensitivity of the contractile apparatus to Ca²⁺. The precise mechanisms underlying this change have been difficult to define, in part because myofilament regulatory proteins contain multiple phosphorylation sites for protein kinase C (PKC), protein kinase A (PKA) and myosin light chain kinase (MLCK), with potentially opposing effects. MLCK increases whereas PKC and PKA have a strong tendency to decrease myofilament Ca²⁺ sensitivity in myocardium. Here we show in mouse cardiac myocytes that PKC-βII can increase Ca²⁺ sensitivity of tension by a similar magnitude to MLCK but via a distinct mechanism. For PKC-βII ³²P-incorporation occurred primarily into cardiac troponin I (cTnI) and functional effects were highly dependent upon mutations in phosphorylation sites of cTnI. Replacement of serines-23/24 (PKA sites) with alanine prevented cross-phosphorylation of these sites, reduced ³²P-incorporation into cTnI by half and resulted in myofilament Ca²⁺ sensitization rather than desensitization in response to PKC-βII. Replacement of three additional sites on cTnI, serines-43/45 and threonine-144, eliminated PKC-βII-mediated Ca²⁺ sensitization and the remaining ³²P-incorporation into cTnI. A preference for PKC-βII phosphorylation of threonine-144 in the intact filament lattice was revealed by differential stable isotope labeling and supported by an analysis of peptide phosphorylation. The results suggest that threonine-144 within the critical inhibitory domain of cTnI represents a novel site of regulation of myofilament Ca²⁺ sensitivity by PKC-βII, with possible implications for chronically stressed or diseased hearts.