Increases in heart rate are accompanied by acceleration of relaxation. This effect is apparent at the single myocyte level and depends on sarcoplasmic reticulum (SR) Ca transport and Ca/calmodulin dependent protein kinase [CaMKII; see R. A. Bassani, A. Mattiazzi, and D. M. Bers.Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H703–H712, 1995]. Because phosphorylation of phospholamban (PLB) by CaMKII can stimulate SR Ca transport, it is a plausible candidate mechanism. We examined this issue using ventricular myocytes isolated from wild-type (WT) mice and those in which the PLB gene was ablated by gene targeting (PLB-KO). During steady-state (SS) stimulation, twitch relaxation and intracellular Ca concentration ([Ca]_i) decline were significantly faster than after a rest in both WT and PLB-KO myocytes. Furthermore, the CaMKII inhibitor KN-93 (1 μM) abolished the stimulation-dependent acceleration of twitch [Ca]_i decline in PLB-KO. This indicates that neither PLB nor its phosphorylation are required for the CaMKII-dependent acceleration of the SS twitch [Ca]_i decline and relaxation. Other quantitative aspects of Ca transport in WT and PLB-KO myocytes were also examined. As expected, the time constant (τ) of [Ca]_i decline during the SS twitch is much faster in PLB-KO than in WT myocytes (112 ± 6 vs. 188 ± 14 ms, P < 0.0001). There was also an increase in SS SR Ca load, based on the change of [Ca]_i during rapid caffeine-induced contractures (CafC) with Na/Ca exchange blocked (565 ± 74 nM for WT, 1118 ± 133 nM for PLB-KO,P < 0.01). Accounting for cytosolic Ca buffering, this implies a 37% increase in SR Ca content. The τ for [Ca]_idecline of the CafC with Na present indicated slower extrusion by Na/Ca exchange in the PLB-KO mouse (2.2 ± 0.2 s in WT vs. 3.2 ± 0.2 s in PLB-KO, P < 0.01), although exchanger protein expression was unchanged. Integrated Ca flux analysis in WT and PLB-KO myocytes, respectively, shows that 90 and 96% of Ca during twitch relaxation is removed by the SR Ca-ATPase, 9 and 3.4% by Na/Ca exchange, and 0.5 and 0.1% by slow mechanisms (mitochondria Ca uniporter and sarcolemmal Ca-ATPase). We conclude that the PLB-KO myocytes retain a CaMKII-dependent acceleration of SS twitch [Ca]_i decline. The PLB-KO (vs. WT) myocytes also have higher SR Ca pump activity, higher SR Ca load, and reduced Na/Ca exchange activity.