Several in vitro studies about age-associated skeletal muscle mitochondrial dysfunction are somewhat conflicting, and this might be related to different normalization procedures. The objective of this study was to normalize the functional and biochemical data per number of mitochondria present in a mitochondrial suspension. Functional and biochemical parameters were obtained in mitochondrial suspensions from murine skeletal muscle of different ages. Mitochondrial respiratory function was polarographically measured using a Clark-type oxygen electrode. Biochemical analyses included determination of citrate synthase (CS) activity and total protein content in the mitochondrial suspension. Electron microscopy analysis of the suspensions allowed calculation of the number of mitochondria per milligram of protein. Our results conclude that advanced age is associated with mitochondrial dysfunction; moreover, from the correlation between morphological and biochemical data, it is evident that CS activity in the mitochondrial suspensions is a more accurate marker of mitochondrial mass than is total protein content.