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Cellular and nuclear uptake of tri-iodothyronine (T3) and thyroxine (T4) was examined using the cultured cell line derived from rat liver, clone 9, and rat hepatoma, dRLH-84. The saturable cellular uptake of T3 and T4 was demonstrated in these cells. First we examined the cell cycle-dependent alteration of thyroid hormone uptake. Cellular T3 uptake was minimal in the early G1 phase and increased in the late G1 phase, reaching a maximal level in the S phase. Alterations in nuclear T3 uptake were in accordance with the changes in cellular T3 uptake. On the other hand, cellular and nuclear T4 uptake was unchanged throughout the cell cycle, suggesting the T3 specificity of the cell cycle-dependent alteration of cellular hormone transport. Next we examined the effect of sodium butyrate on the cellular transport of thyroid hormones. After treatment with 5 mM sodium butyrate, cellular and nuclear uptake of T3 was increased, reaching a maximal level (four- to sevenfold increase) after 48 h. When cells were incubated for 48 h with various concentrations of sodium butyrate, T3 uptake was enhanced by 1 mM sodium butyrate, reaching a maximal level with 5 mM. Although cellular T4 uptake was also increased after treatment with sodium butyrate, the degree and time-course of the increase were different from those of T3. The maximal increase in cellular T4 uptake (two- to threefold increase) was attained 20 h after treatment. Despite the increase in cellular T4 uptake, nuclear T4 uptake was decreased after treatment with sodium butyrate. For both T3 and T4, the enhanced cellular uptake was due to the increased Vmax without changes in the Michaelis-Menten constant. These data indicate that cellular transport of T4 is different from that of T3 in rat hepatic cells.