The mechanism of T3 entry into cells was studied in undifferentiated NB41A3 neuroblasts and after differentiation with 0.5 mM sodium butyrate. In undifferentiated neuroblasts, cell uptake of labeled L-T3 at 2 h was reduced to 43% of the control value by excess L-T3, whereas only 5.9% of D-T3 uptake was saturable. After incubation of intact cells with labeled hormone, the nuclei contained 3.8% of the total cellular L-T3 and 4.3% of D-T3. Thus, L-T3 nuclear uptake was 3 times higher than D-T3. Kinetic analysis of the initial rate of uptake of L-T3 by the cells gave a Km of 1.25 nM and a maximum velocity of 2.38 fmol/min.10(6) cells. The initial rate of D-T3 uptake was not saturable. Inhibitors of ATP production (antimycin and oligomycin) as well as monodansylcadaverine virtually abolished saturable cell uptake and decreased nuclear uptake more than total cell uptake, suggesting that the saturable component of uptake into the cells is the major source of nuclear L-T3. After differentiation by butyrate, both cell uptake and nuclear uptake of L-T3 increased. The apparent affinity (Ka) of the nuclear T3 receptors was determined in intact cells and compared with the Ka measured with isolated nuclei. In undifferentiated cells, the apparent Ka was 3-fold higher than the true Ka, presumably due to a step-up in the free T3 concentration in the cytosol compared to that in the incubation medium. After butyrate exposure, the apparent nuclear Ka was decreased to less than 1.5 times the control value, but Ka in isolated nuclei was unchanged. The number of nuclear receptors, however, was increased by butyrate in both intact cell and isolated nuclei experiments. These results indicate that butyrate exerts separate effects on the number of nuclear receptors and saturable T3 transport in mouse neuroblasts.