The lipid domains of the cell membrane are believed to be one of the sites where biguanides exert their antihyperglycemic effect. We have examined the effects of metformin on the membrane fluidity of intact erythrocytes in vivo and in vitro. Membrane fluidity was measured by monitoring changes in the anisotropy of the fluorescent probe 6-antroyloxystearic acid (6-AS). The erythrocyte membranes from patients with non-insulin dependent diabetes mellitus treated with metformin were more fluid than those from non-insulin dependent diabetes mellitus patients treated by diet or healthy controls. There was no correlation between membrane fluidity and the plasma lipids or the parameters of metabolic control, suggesting that the high fluidity is an effect of metformin itself. Incubation of erythrocytes from healthy controls and diabetic patients treated by diet or glibenclamide with metformin in vitro confirmed that metformin increases the fluidity of erythrocyte membranes. In vitro metformin did not alter the fluidity of membranes from diabetic patients treated with metformin, perhaps because the basal high fluidity due to their in vivo interaction with plasma metformin could be increased no further. Since insulin appears to be required for the antihyperglycemic effect of metformin, the effect of insulin on membrane fluidity was also evaluated. Insulin generally had a small fluidizing effect on erythrocytes in vitro. The fluidizing action of both insulin and metformin could represent a membrane event common to the hormone and drug leading to additive or synergistic effects in vivo.