An ideal assay of adenine nucleotides, lactate and pyruvate in the extract of rat freeze-clamped muscle was achieved. Electromicroscopy showed that the cell membrane was destroyed by powdering the muscle in liquid N_2 and then homogenizing for 1 min. An additien of ultrasonic treatment for 1 min could thoroughly break the subcellular structures and lead to a remarkable increase in ATP and total adenine nucleotides with a little change of lactate and pyruvate. The prolongation of the treatment for two more minutes, however, could result in the breakdown of ATP.