(1)H magnetic resonance spectroscopic imaging (MRSI) was used to investigate the effect of orientation on spectral characteristics of trimethyl ammonium (TMA) in human muscle at rest. Four different muscles in the healthy calf were studied: soleus, gastrocnemius, tibial posterior and anterior. The data demonstrate that muscle orientation can profoundly change apparent spectral characteristics of proton metabolites. In particular, muscle orientation can cause concerted changes in the spectral pattern of TMA/methyl (tCr) and methylene (Cr2) protons of creatine for a given muscle, a switch of TMA/tCr spectral patterns among different muscles and changes in the T(2) of TMA. A significant correlation was detected between TMA/tCr peaks and the Cr2 peak splitting (r=.62, P<.001). In vivo (1)H MRSI has the potential to simultaneously evaluate the orientation of muscle fibers and biochemical changes induced by a disease process or physiological activity.