The purpose of this study is to determine the effects of brief rapid pacing (RP; ~200-240 beats/min for ~5 min) on contractile function in ventricular myocytes. RP was followed by a sustained inhibition of peak systolic cell shortening (44 ± 4%) that was not due to changes in diastolic cell length, membrane voltage, or L-type Ca²⁺ current (I_Ca,L). During RP, baseline and peak intracellular Ca²⁺ concentration ([Ca²⁺]_i) increased markedly. After RP, Ca²⁺ transients were similar to control. The effects of RP on cell shortening were not prevented by 1 µM calpain inhibitor I, 25 µM L-N⁵-(1-iminoethyl)-orthinthine, or 100 µM N^G-monomethyl-L-arginine. However, RP-induced inhibition of cell shortening was prevented by lowering extracellular [Ca²⁺] (0.5 mM) during RP or exposure to chelerythrine (2-4 µM), a protein kinase C (PKC) inhibitor, or LY379196 (30 nM), a selective inhibitor of PKC-. Exposure to phorbol ester (200 nM phorbol 12-myristate 13-acetate) inhibited cell shortening (46 ± 7%). Western blots indicated that cat myocytes express PKC-, -, and - as well as PKC-. These findings suggest that brief RP of ventricular myocytes depresses contractility at the myofilament level via Ca²⁺/PKC-dependent signaling. These findings may provide insight into the mechanisms of contractile dysfunction that follow paroxysmal tachyarrhythmias.