The viscosity of the phospholipid bilayer and the wobbling angle of the phospholipid molecules of mitochondrial membranes and plasma membranes from bullfrog myocardium were measured with a nanosecond time-resolved fluorometer using pulsed excitation of a hydrophobic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). The mean +/- S.D. in the viscosity of the mitochondrial and plasma membranes was 0.54 +/- 0.9 and 0.37 +/- 0.03 P, respectively, at 30 degrees C. The wobbling angle of phospholipid molecules was 42 +/- 1 and 47 +/- 1 degree, respectively. Cholesterol content was lower in mitochondria (6.4 micrograms/mg protein) than in plasma membranes (43.7 micrograms/mg protein) but phosphatidylethanolamine concentration was higher in mitochondria (31.8%) than in plasma membranes (27.3%). Cardiolipin was contained only in mitochondria. The results of these lipid analyses appear consistent with the measurements of membrane viscosity and phospholipid wobbling angle. When the results are compared with those from a previous study on the erythrocyte membranes from bullfrogs, viscosity is found to increase in the order mitochondrial membranes less than plasma membranes less than erythrocyte membranes. The complex requirements of biomembranes of organelles performing different functions appear to be met by the particular dynamic microstructure of the biomembrane. The effect of membrane viscosity on oxygen diffusion through membranes is discussed.