Insulin receptor internalization in response to insulin incubation (down-regulation) has been shown to occur in human erythrocytes as well as in human erythrocyte ghosts. It is also known that changes in cell membrane events can be detected with electron paramagnetic resonance (EPR) spectroscopy using spin labels. In the present study, changes in erythrocyte membrane order during down regulation as measured by the 2T'II parameter were investigated using EPR. The spin label, 5-nitroxystearate, which inserts into the lipid bilayer of cell membranes, was used. Changes in 2T'II at 37 degrees C were followed over a 3 h time period. A transient decrease in erythrocyte membrane order began within 30 min of the start of insulin incubation and reached a minimum level of 52.5 Gauss (G) within 90 min. This represented nearly a 2 G decrease from the zero incubation time value. Membrane order returned to the initial value by 2.5 h. These time-related changes in membrane order corresponded well with the insulin receptor internalization process as followed by surface binding assays. Surface insulin binding began to decrease within 30 min of the start of insulin incubation, and was reduced to 30% of control values within 2 h. Similar correlations between membrane order and receptor internalization were observed at 23 degrees C. Erythrocytes incubated with denatured insulin, and ATP-depleted erythrocytes incubated with native insulin, did not down-regulate their insulin receptors. Under these conditions, these erythrocytes also did not exhibit the transient decreases in membrane order. These findings are consistent with the hypothesis that an increase in membrane disorder is part of the mechanism of insulin receptor down-regulation.