SUMMARY: The binding of 125I-labelled thyroxine and tri-iodothyronine to a 100000 g supernatant fraction (cytosol) from homogenates of porcine anterior pituitary lobes was examined. The kinetics of thyroxine binding were studied and the dissociation rate-constant observed at 4 °C was 2·4 × 10−4 s−1. Equilibrium constants of relatively high affinity for both iodothyronine hormones were obtained from Scatchard plots of dose-response data, and were found to be 0·4 × 109 m−1 for tri-iodothyronine and 1·4 × 109 m−1 for thyroxine. Bound [125I]thyroxine could be virtually completely displaced by unlabelled thyroxine, but only partially by unlabelled tri-iodothyronine. From these relative potency experiments it is tentatively suggested that a thyroxine-specific site of low affinity is present in the cytosol. A protein component is probably involved in thyroxine binding, since a decrease in bound hormone was seen only after treatment with proteolytic enzymes. The thyroxine-binding capacities of anterior pituitary cytosol and porcine serum were similar, and were two or three times greater than the binding capacities of cytosol fractions from either posterior pituitary or brain frontal lobe. These results suggest that thyroxine binding in the anterior pituitary cytosol is unlikely to have resulted solely from contamination by serum thyroxine-binding proteins. Further evidence for this belief was provided by thin-layer chromatographic separation of labelled thyroxine bound to either cytosol or to serum proteins. The possible relevance of these studies to the feedback regulation of thyrotrophin secretion is discussed.