The manner of inhibition of thyroid I- accumulation by perchlorate (CIO4-) and thiocyanate (SCN-) was studied using a newly developed biological model of the I- transport system. CIO4- inhibited I- accumulation in phospholipid vesicles made from thyroid plasma membrane and soybean phospholipids by decreasing Na+-dependent I- influx. The anion did not at all induce I- leakage from the vesicles. On the basis of Lineweaver-Burk plot analysis, it did not change Vmax for I- concentration. These results suggest that CIO4- is a competitive inhibitor of thyroid I- transport. In contrast, SCN- did increase I- leakage from the phospholipid vesicles to diminish I- accumulation. This anion might cause only slight depression of Na+-dependent I- entry, if any. The results do not support the idea that SCN- may be a competitive inhibitor, in spite of the fact that the anion did not change Vmax for I- transport on the basis of Lineweaver-Burk plot analysis.