Creatine kinase (CK) has been implicated in the maintenance of skeletal muscle intracellular energy supply via its ATP buffering capacity. We examined the postnatal expression of CK activity and isoform phenotype in four skeletal muscles [diaphragm (DIA), intercostal (IC), external abdominal oblique (EAO), and the soleus (SOL)] of the rat. Moreover, we correlated CK activity during development with postnatal changes in myosin heavy chain (MHC) phenotype, the latter an index of relative changes in the energetic demands of muscle contractile proteins. CK activity was lowest in the immediate newborn period and increased in all muscles during postnatal development; the highest levels of CK activity were observed in the adult IC and EAO. CK activity did relate to the MHC phenotype as indexed by the ratio of adult MHC isoform content (slow + IIa + IIx + IIb) to developmental MHC isoform content (slow + neonatal; r2 = 0.93, p < 0.001). Stepwise regression revealed that type IIb MHC expression alone accounted for 79% of the developmental variance in CK activity. We conclude that CK activity increases during postnatal development in a muscle specific fashion and relates to the energetic demands of the muscle contractile proteins as reflected by MHC isoform composition. We speculate that the role of CK as an energy buffer is greatest in muscles expressing the IIb MHC isoform.