We have recently described the transferrin-mediated uptake and release of iron by BeWo cells [van der Ende, du Maine, Simmons, Schwartz & Strous (1987) J. Biol. Chem. 262, 8910-8916]. We now extend our studies of the mechanisms responsible for uptake and release of iron by these cells. Following preloading, 59Fe release was maximal (about 12%) after about 4 h. Replacement of the extracellular medium with an equal volume of fresh medium either prior to or following the time at which equilibrium was reached further stimulated 59Fe release. Both the rate and maximum amount of iron release decreased if longer loading times were used. Preincubation of BeWo cells for 15 min with 10 mM-sodium cyanide and 50 mM-2-deoxyglucose prior to the determination of 59Fe release did not alter the amount released into medium (which did not contain a high-affinity iron chelator). However, under these conditions, the uptake of 59Fe was dramatically inhibited as a result of prolongation of the transferrin-transferrin-receptor complex recycling time. These results demonstrate that the release of iron from BeWo cells is independent of cellular ATP levels, whereas iron uptake is ATP-dependent. Rates of both 59Fe release and 59Fe uptake were temperature-dependent. Analysis of these data via an Arrhenius plot suggests a single rate-limiting step for the release and uptake processes between 0 and 37°C. The apparent energies of activation of these processes are very similar (approx. 59.0 kJ/mol for iron release and 50.6 kJ/mol for iron uptake), which raises the possibility that the release and uptake of iron share a common thermodynamically rate-limiting step. Possible mechanisms involved in iron release out of the cell and out of the endosome are discussed.