Different levels of calorie restriction (CR) (125, 85, 50, or 40 kcal/wk for 1, 3, and 6 months) were examined in mice by using the paradigm of Weindruch and colleagues. Lean and total body mass increased on 125 and 85 kcal/wk, but there was negligible growth on low-energy intake. There was no CR-induced reduction in either daily activity or mass-specific metabolic rate. There was no CR-effect on in vitro reactive oxygen species production by liver or muscle mitochondria at 3 months, but after 6 months the effect was significantly reduced in liver mitochondria from 40 kcal/wk mice compared to 125 kcal/wk mice. Changes in the fatty acid composition of phospholipids from liver, kidneys, heart, brain, and skeletal muscle were observed following 1 month of CR. Copyright 2006 by The Gerontological Society of America.