Chronocoulometry was used to characterize the fluidity and lateral diffusion coefficient of supported phospholipid bilayer assemblies. The bilayers were formed on the inner surfaces of the microporous template films of aluminum oxide on gold electrodes. The lipid monolayers were formed by adsorption and fusion of phospholipid vesicles on alkylated oxide surfaces. Octadecyltrichlorosilane (OTS) was used in the initial alkylation step. The surface concentration of the lipids in monolayer assemblies was measured by a radioactive assay method. Surface densities corresponding to 48 +/- 10 A2/molecule (DPPC) and 56 +/- 11 A2/molecule (DMPC) were obtained (for exposure times > 120 min) independent of the temperature of the vesicle's fusion (below or above chain-melting transition). Octadecylviologen (C18MV2+) was used as an electroactive probe species. Its limiting lateral diffusion coefficient in DMPC monolayers was 5 x 10(-8) cm2/s, measured as C18MV2+ mole fraction extrapolated to 0 decreasing linearly from 20 to below 1 mol%. Linear Arrhenius plots for C18MV2+ diffusion in DMPC monolayers were obtained with slopes of approximately 40 kJ/mol between 18 and 45 degrees C, demonstrating homogeneity and fluidity of the lipid monolayers. Chronocoulometry was also used to obtain lateral diffusion coefficient of ubiquinone in DMPC/OTS bilayers. A value of 1.9 x 10(-8) cm2/s at 30 degrees C was obtained.