Several biochemical components of the white portion of the gastrocnemius (WGM), plantaris (PM), and soleus (SM) muscles of the rat and middle gluteal (MGM) muscle of the horse were compared based on wet and dry weight, protein, and total creatine concentrations ([TCr]). The water content was similar for the rat hindlimb muscles, however, the concentrations of protein, ATP, phosphocreatine (PCr), creatine, and glycogen ranked as SM less than PM less than WGM for all reference bases except total creatine. In contrast, concentrations of ATP, creatine, and PCr were similar in all muscles studied when expressed as ratios of [TCr]. Horse MGM had the lowest percent of water and protein per gram wet or dry weight but highest glycogen concentration of the muscles studied, irrespective of the reference base used to express concentrations. Coefficients of variation were lowest when muscle constituents were related to [TCr]. It is concluded that expressing muscle constituents relative to total creatine results in the smallest variation and is a good method for making comparisons between muscles of similar fiber composition. However, essential information concerning different types of muscle may be lost when this reference base is used.