Macrophages in the vessel wall secrete high levels of apolipoprotein E (apoE). Cholesterol efflux from macrophages to apoE has been shown to decrease foam cell formation and prevent atherosclerosis. An apoE molecule can mediate cholesterol efflux from the macrophage that originally secreted it (autocrine effect) or from surrounding macrophages (paracrine effect). Traditional methodologies have not been able to separate these serial effects. The novel methodology presented here was developed to separate autocrine and paracrine effects by using a simple mathematical model to interpret the effects of dilution on apoE-mediated cholesterol efflux. Our results show that, at very dilute concentrations, the paracrine effect of apoE is not evident and the autocrine effect becomes the dominant mediator of efflux. However, at saturating concentrations, paracrine apoE causes 80-90% of the apoE-mediated cholesterol efflux, whereas autocrine apoE is responsible for the remaining 10-20%. These results suggest that the relative importance of autocrine and paracrine apoE depends on the size of the local distribution volume, a factor not considered in previous in vitro studies of apoE function. Furthermore, autocrine effects of apoE could be critical in the prevention of foam cell formation in vivo. This novel methodology may be applicable to other types of mixed autocrine/paracrine systems, such as signal transduction systems.