First, an 80% 59Fe-saturated transferrin solution was exposed to reticulocytes to reduce its iron content. Iron-donating activity of the supernatant was subsequently compared to that of a fresh preparation equal in iron and transferrin concentration; it was found significantly reduced, but gradually improving in presence of 10(-3)M citrate. Second, two transferrin solutions saturated with 59FeCl3 or 56FeCl3 were exposed to reticulocytes and resaturated adding the opposite iron species. These preparations were injected into rats and the 59Fe plasma disappearance rate was followed; the 59Fe added after reticulocyte exposure was cleared more rapidly. It is concluded that citrate ions mediate interbinding site iron exchange. Further, iron distribution among binding sites is important for tissue iron delivery.