The mechanism of inhibition of I- accumulation in the thyroid by valinomycin was investigated on a biological model. Phospholipid vesicles (P-vesicles) capable of Na+-dependent I- accumulation were reconstituted with thyroid plasma membrane (PM). In P-vesicles internally loaded with K+, Na+-dependant I- accumulation was observed in the presence of external Na+. The accumulated I- was discharged from the vesicles by addition of valinomycin. In P-vesicles with internal choline+, Na+-dependent I- accumulation also occurred, but I- discharge was not induced by the addition of valinomycin. P- vesicles without a PM component readily accumulated I- in the presence of both external K+ and valinomycin. P-vesicles with thyroid PM also accumulated I- rapidly in the presence of external K+ upon the addition of valinomycin; however, the accumulated I- was significantly discharged when the vesicles were loaded internally with Na+. A significant I- discharge was not observed in the vesicles with or without thyroid PM when they were loaded with choline+ instead of Na+. Valinomycin-induced I- uptake without leakage was observed in both Na+- and choline+-loaded P-vesicles containing liver PM instead of thyroid PM. These results indicate that valinomycin may inhibit I- accumulation in thyroid cells according to the following mechanism: 1) valinomycin, a K+ carrier, induces K+ efflux to form a gradient of electrical potential with a negative charge within the cells, and 2) I- is subsequently driven out down the gradient through the phospholipid bilayer. Na+-dependent I- transport may not be impaired by valinomycin.