By means of a micropuncture technique and microgel electrophoresis the luminal content of single rat thyroid follicles were analyzed during PTU treatment for 4 weeks. The relative amount of aggregates of thyroglobulin (i.e. the 27S iodoprotein and larger complex) was reduced from 15 per cent to approximately 3 per cent after 48 h. The aggregates were practically absent after 2 weeks. The thyroglobulin fraction was the predominant colloid constituent. The protein concentration of the colloid was not changed at the end of the period of PTU treatment (8.9 g/100 ml). It is concluded that PTU inhibits the aggregation of thyroglobulin to a larger complex. The results further indicate that the aggregates of thyroglobulin are preferentially localized to the periphery of the lumen in the larger follicles.