After a preliminary study showed conclusively that in vitro pineal cells avidly take up thyroid hormones but not iodide, a series of tissue slice studies using 125I-thyroxine were conducted to investigate the mechanism of thyroxine (T4) uptake in pineal cells. T4 uptake was unaffected by the presence or absence of glucose and cofactors, nor was uptake inhibited by cyanide. No binding capacity for T4 could be demonstrated, and the percent uptake of labelled T4 actually increased with T4 concentration up to a certain level. Uptake of the thyroid hormone was quite rapid. The temperature coefficient was determined for the uptake, and was found to fall in the range of values reported for diffusion mechanisms. On the basis of these findings, we concluded that energy dependent mechanisms of transport could be eliminated, and that some sort of mediated diffusion was the most likely mechanism for T4 transport.