Exposure of human erythrocytes to elevated intracellular calcium causes alterations in cell shape and stimulates shedding of the cell membrane in the form of microvesicles. We hypothesized that both the shape transition and microvesiculation are influenced by microscopic membrane physical properties such as lipid order. To test this hypothesis, membrane properties were manipulated by varying the experimental temperature, membrane cholesterol content, and the internal ionic environment. Changes in membrane order were assessed using steady-state fluorescence spectroscopy with an environment-sensitive probe, laurdan. Our observations led us to the following conclusions: 1) the modest temperature dependence of membrane structure observed with laurdan is shifted to lower temperatures and becomes more cooperative upon removal of membrane cholesterol, 2) the calcium-induced shape change observed in erythrocytes requires a decrease in membrane order, 3) the influence of membrane order is not limited to shape transitions induced only by calcium, and 4) decreased order is also a permissive factor for microvesicle shedding. Our data suggest that while the mechansims that regulate the shape transition and the release of microvesicles are different, they both require a state of membrane disorder.