The active accumulation of I- in the thyroid gland is mediated by the Na(+)-I- symporter and driven by the Na+ gradient generated by the Na+/K(+)-ATPase. Thyrotropin (TSH) stimulates thyroidal I- accumulation. Rat thyroid-derived FRTL-5 cells require TSH to accumulate I-. TSH withdrawal for over 7 days results in complete loss of Na(+)-I-symport activity in these cells [Weiss, S. J., Philp, N. J. and Grollman, E. F. (1984) Endocrinology 114, 1090-1098]. Surprisingly, membrane vesicles prepared from FRTL-5 cells maintained in TSH-free medium [TSH(-)cells]accumulate I-, suggesting that the absence of Na(+)-I- symport activity in TSH(-) cells cannot be due solely to a decrease in the biosynthesis of either the symporter or a putative activating factor. This finding indicates that the Na(+)-I- symporter is present, probably in an inactive state, in TSH(-) cells despite their lack of Na(+)-I- symport activity. Na(+)-I- symport activity in thyroid membrane vesicles is enhanced when conditions for vesicle preparation favor proteolysis. Subcellular fractionation studies in both TSH(+) and TSH(-) cells show that Na(+)-I- symport activity is mostly associated with fractions enriched in plasma membrane rather than in intracellular membranes, suggesting that the Na(+)-I- symporter may constitutively reside in the plasma membrane and may be activated by TSH.