To investigate the uptake of triiodothyronine sulfate (T3S) and its effect on thyrotropin-releasing hormone (TRH)-induced thyrotropin (TSH) secretion, anterior pituitary cells were isolated from euthyroid rats and cultured for 3 days in medium containing 10% fetal calf serum. Incubation was performed at 37 degrees C in medium containing 0.5% bovine serum albumin (BSA). Exposure of the pituitary cells to TRH (0.1 mu mol/L) for 2 hours stimulated TSH secretion by 176%. This effect was reduced by approximately 45% after a 2-hour preincubation with T-3 (0.001 to 1 mu mol/L). A significant inhibitory effect of T3S on TRH-induced TSH release was only observed at a concentration of 1 mu mol/L. The uptake of [I-125]T-3 after 1 hour of incubation was reduced by 40% +/- 4% (P < .001) by simultaneous addition of 10 nmol/L unlabeled T-3, whereas 1 mu mol/L T3S was required to obtain a reduction of the [I-125]T-3 uptake by 34% +/- 2% (P < .001). The amount of T-3 present in the unlabeled T3S preparation was 0.25% as determined by radioimmunoassay. When pituitary cells were incubated for 1 hour with [I-125]T3S or [I-125]T-3 (both 50,000 cpm/0.25 mL), the uptake of [I-125]T3S expressed as a percentage of the dose was 0.04% +/- 0.02% (mean +/- SE, n = 4), whereas that of [I-125]T-3 amounted to 3.0% +/- 0.4% (n = 4). In contrast, when hepatocytes were incubated for 1 hour with [I-125]T3S, the uptake amounted to 5.1% +/- 0.8% (n = 9), whereas that of [I-125]T-3 was 22.1% +/- 1.7% (n = 9). Furthermore, [I-125]T3S was as rapidly deiodinated (iodide production, 14.9% +/- 2.6%; n = 9) as [I-125]T-3 (12.1% +/- 0.8%, n = 9) by hepatocytes. It is concluded that (1) T3S is poorly taken up by pituitary cells, and (2) the suppressive effect of high concentrations of T3S on TRH-induced TSH secretion and on [I-125]T-3 uptake can be explained by slight contamination with T-3. Thus, it appears that T3S has only a minor biological effect, if any, on the pituitary. Copyright (C) 1994 hy W.B. Saunders Company