Thyroglobulin (Tg), the precursor of thyroid hormones, follows a unique secretion, storage, and recapture pathway. The first steps of recapture were studied by investigating the binding of 125I-labeled Tg on the apical surface of inside-out follicles and its internalization. The selectivity of the process was assessed by using molecules other than Tg and/or nonthyroid cells. Tg binding to the apical surface of thyroid inside-out follicles is selective relative to the binding of other molecules. It increases sharply over pH 8.0 and occurs through specific sites of moderately high affinity (Kd = approximately 200 nM; 2 x 10(4) sites/cells). At pH < 8.0 it occurs through numerous sites of very low affinity considered nonspecific. Endocytosis, although weak under our conditions, increases at pH 8.0 concomitantly with binding. Over pH 8.2, however, some inhibition occurs. Surprisingly, Tg binding and endocytosis are not tissue specific, as they showed the same properties on thyroid inside-out follicles and Madin-Darby canine kidney or Chinese hamster ovary cells. Thus, a selective uptake of Tg mediates its recapture by thyroid cells. This selectivity is an intrinsic Tg property, not dependent on the thyrocyte apical surface, as it was observed with Madin-Darby canine kidney and Chinese hamster ovary cells. Given the pH effect observed, we suggest that Tg binding is a regulated phenomenon and that, through luminal pH control, it can vary from a basal level at neutral pH to a stimulated level over pH 8.0.