Methods are described for the determination of glycogen, glycolytic intermediates, and high-energy phosphates in muscle biopsy samples. Initial freezedrying of samples and extraction of metabolites with relatively weak acid are preferred. Normal values in muscle are similar to those found by other workers. Variation in muscle content of ATP, ATP + ADP + AMP, phosphorylcreatine (PC), creatine (Cr), PC + Cr, and glycogen, between legs, between sites on the same muscle, or as a result of error introduced during analysis, was small compared with the between-individuals variance. The importance of the different sources of variance on taking a biopsy is discussed.