The effects of creatine (Cr) absence in skeletal muscle caused by a deletion of guanidinoacetate methyltransferase (GAMT) was studied in a knockout mouse model by in vivo ³¹P magnetic resonance (MR) spectroscopy. ³¹P MR spectra of hind leg muscle of GAMT deficient (GAMT-/-) mice showed no phosphocreatine (PCr) signal but instead the normally not present signal of phosphorylated guanidinoacetate (PGua), the immediate precursor of Cr. Tissue pH did not differ between wild type (WT) and GAMT-/- mice while relative inorganic phosphate (Pi) levels were increased in the latter. During ischemia, PGua was metabolically active in GAMT-/-mice and decreased at a rate comparable to the decrease of PCr in WT mice. The recovery rate of PGua after ischemia, however, was reduced compared to PCr in WT mice. Saturation transfer measurements revealed no detectable flux from PGua to -ATP, indicating severely reduced enzyme kinetics. Supplementation of Cr resulted in a rapid increase in PCr signal intensity until only this resonance was visible, along with a reduction in relative Pi values. PGua recovery rate after ischemia, however, did not change. Our results show that despite the absence of Cr, GAMT-/- mice can cope with mild ischemic stress by using PGua for high energy phosphoryl transfer. The reduced affinity of creatine kinase (CK) for (P)Gua only becomes apparent during recovery from ischemia. It is argued that absence of Cr causes the higher relative Pi concentration also observed in animals lacking muscle CK, indicating an important role of the CK system in Pi homeostasis.