There are changes in the mixed function oxidase enzymatic activities of rat hepatic microsomal membranes with age. However, the protein components of the mixed function oxidase system do not appear to change with age. The purpose of this study was to detect possible changes in the fluidity of the lipid component of the microsomal membrane with age. Hepatic microsomes were isolated by differential centrifugation from uninduced, male CFN rats aged 3, 12 and 26 mo. The microsomal membrane fluidity was measured using electron paramagnetic resonance after incorporation of a 5-nitroxide stearic acid spin label into the membrane. The order parameter S decreased with age from 0.586 +/- 0.003 (3 mo) to 0.581 +/- 0.002 (12 mo) to 0.569 +/- 0.003 (26 mo) at 30 degrees C. This indicated an increase in membrane fluidity with age. In membranes labeled with the 16-nitroxide stearic acid, a similar increase in membrane fluidity with age was observed. The order parameter of microsomal membranes from 3 and 26 mo rats was measured over the temperature range 10 degrees to 31 degrees C in steps of 0.9 degrees C. A plot of the log of S versus the reciprocal temperature revealed a phase transition at 24 degrees C in membranes from 26 mo rats, but no phase transition was observed in 3 mo old rats in this temperature range. The change in fluidity of the hepatic microsomal membrane with age may account for some of the observed changes in membrane-bound mixed function oxidase activities with age.