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Because it has been suggested that decreased activity at the erythrocyte sodium pump might be the cause of age-related decreases in basal oxygen consumption, we have examined age-associated changes in Na+,K+-ATPase activity in red cell membranes. The initial portion of this study was directed toward elucidating possible methodological pitfalls in membrane preparation which might account for some of the variable results reported in prior erythrocyte Na+,K+-ATPase studies. We found that two of four red cell membrane fractions have substantial Mg2+-ATPase activity and contribute a significant portion of total membrane protein. As these two fractions contain little Na+,K+-ATPase activity their contamination of the other two fractions could cause significant variation in measured Na+,K+-ATPase activity. Additionally, we found that meal feeding raised Na+,K+-ATPase activity necessitating that measurements be made in the fasting state. With these methodological variables controlled, we found only a 10.8% coefficient of variation between fasting samples obtained on separate days in eight subjects. Using this methodology, we observed no correlation of Na+,K+-ATPase specific activity with age in males, and only a weak correlation in females who showed decreasing Na+,K+-ATPase specific activity occurring with advancing age. These observations suggest that changes in erythrocyte Na+,K+-ATPase activity do not cause the age-related fall in basal oxygen consumption.