To understand better the role of the creatine kinase (CK)/phosphocreatine system in muscle bioenergetics, a series of mouse mutants with subnormal muscle CK (M-CK) expression has been generated. Here we compare the phenotypes of mice deficient in M-CK (M-CK-/-) and M-CK leaky-mutant mice, which carry a targeted insertion of a hygromycin B-poly(A) resistance cassette in the second M-CK intron. Mice homozygous for this M-CK allele (M-CKI/I) have a 3-fold reduction of dimeric muscle CK enzyme activity, whereas compound heterozygotes with the null M-CK allele (M-CKI/-) display a 6-fold reduction. Unlike M-CK-/- mice, these mutants have no increased glycogen content or glycogen consumption in their fast fibers. The intermyofibrillar mitochondrial volume of these fibers is also normal, suggesting that energy transport via the CK/phosphocreatine system may function at low myofibrillar M-band CK levels. Conversely, the flux of energy through the CK reaction is still not visible by means of 31P NMR spectroscopy, indicating that relatively high levels of M-CK expression (> 34% of normal) are required to generate CK fluxes detectable by this technique. The ability of muscles to perform burst activity is also subnormal and closely correlates with the level of M-CK expression.