The effects of benzyl alcohol on cell shape, hemileaflet lipid fluidity and membrane rheology of human red blood cells were studied. Membrane fluidity was assessed by determining the fluorescence anisotropy of permeant probes (1,6-diphenyl-1,3,5-hexatriene,12-(9-anthroyloxy)stearate, 2-(9-anthroyloxy)stearate) and a new impermeant probe (N-stachyosylsuccinic acid dihydrazide-2-(9-anthroyloxy)stearate). Measurements made on intact red blood cells reflected primarily the outer leaflet fluidity while measurements made on red blood cells ghosts reflected the fluidity of both leaflets. Membrane viscoelasticity was determined by micropipette aspiration. Treatment of intact red blood cells with benzyl alcohol up to 50 mM caused progressive stomatocytic shape change but no change in membrane viscoelasticity, 1,6-diphenyl-1,3,5-hexatriene anisotropy or stachyosyldihydrazide-2(9-anthroyloxy)stearate correlation time; similar treatment of leaky ghosts yielded decreases in 1,6-diphenyl-1,3,5-hexatriene anisotropy and stachyosyldihydrazide-2(9-anthroyloxy)stearate correlation time. With benzyl alcohol above 50-60 mM, intact red blood cells became echinocytic, and decreases in 1,6-diphenyl-1,3,5-hexatriene anisotropy and stachyosyldihydrazide-2(9-anthroyloxy)stearate correlation time occurred in both intact cells and ghosts; there was no change in membrane viscoelasticity. These results indicate that benzyl alcohol up to 50 mM affects primarily the inner leaflet of the red blood cell membrane and that higher concentrations affect both leaflets. These increases in membrane fluidity are not associated with changes in membrane viscoelasticity. This study illustrates the use of fluorescence techniques to monitor specifically the lipid fluidity of each hemileaflet of the erythrocyte membrane.