P21 activated kinase-1 (Pak1) is a serine-threonine kinase, which associates with and activates PP2A in adult ventricular myocytes and thereby induces increased Ca²⁺-sensitivity of skinned-fiber tension development mediated by dephosphorylation of myofilament proteins (Ke et al. 2004). In the present study we test the hypothesis that activation of Pak1 also moderates cardiac contractility through the regulation of intracellular Ca²⁺ fluxes. Our evidence demonstrated that field-stimulated [Ca²⁺]_i-transient amplitude and extent of cell shortening were not different in myocytes expressing constitutively-active Pak1 compared to controls expressing LacZ, however the time to peak shortening was significantly faster and the rate of [Ca²⁺]_i decay and time of re-lengthening were slower. The caffeine-releasable sarcoplasmic reticulum (SR) Ca²⁺ content was not different in CA-Pak1 myocytes compared to controls, nor was fractional-release. Isoproterenol (ISO) application revealed a significantly blunted increase in the [Ca²⁺]_i-transient amplitude in CA-Pak1 myocytes as well as a slowed rate of [Ca²⁺]_i decay, increased SR Ca²⁺ content and increased cell shortening. We found no significant change in CA-Pak1 phospholamban phosphorylation at either Ser16 or Thr17. Analysis of cTnI revealed a significant reduction in CA-Pak1 phospho-species that are primarily attributable to Ser 23/24. Non-stimulated, spontaneous SR Ca²⁺ release sparks were significantly smaller in amplitude in CA-Pak1 than in LacZ myocytes. Spontaneous Ca²⁺ waves resulting from SR Ca²⁺ overload propagated significantly slower in CA-Pak1 myocytes. Our data indicate CA-Pak1 expression has significant effects on ventricular myocyte contractility through altered myofilament Ca²⁺ sensitivity and through modification of the intracellular [Ca²⁺]_i-transient.