The development of tight junctions in fetal rat thyroid from the sixteenth to the twentieth days of gestation was examined with conventional ultrastructural methods and freeze-fracture preparations. These results were compared with those obtained using lanthanum hydroxide and horseradish peroxidase (HRP) tracers. Tight junctions appear to arise on the plasma membranes of fetal thyroid cells by the aggregation and fusion of linear particle chains which appear at several discrete sites on the plasma membrane of developing follicular cells. Tracer studies show that they are effective barriers to the passage of HRP from the outset, are freely penetrated by La3+ at the sixteenth and seventeenth days of gestation, but progressively lose permeability to La3+ from the seventeenth to twentieth days of gestation. However, freeze-fracture observations suggest that La3+ must penetrate into the follicular lumen through the tight junction elements, for the follicular lumen, when it appears, is always completely surrounded by a continuous though sometimes rudimentary meshwork of tight junction elements. The results suggest that the tight junction forms an effective barrier to the passage of large macromolecules, e.g. thyroglobulin, from very early stages in its development. The La3+ results suggest that decreased resistance of the intercellular pathway, possibly related to the development of transepithelial potentials, may occur during this period in development.