GH1 cells are a clonal strain of rat pituitary tumor cells which synthesize GH and PRL. We have previously demonstrated that these cells respond to physiological concentrations of L-T3 and L-T4 when cultured with medium supplemented with thyroidectomized calf serum to achieve a thyroid hormone-depleted state under cell culture conditions. In this study, we describe a method to deplete euthyroid calf serum of L-T3 and L-T4 using an anion exchange resin. We demonstrate that the procedure only minimally alters the low molecular weight anion components of the serum and does not change the total protein content or the electrophoretic pattern of serum proteins. Moreover, we show that euthyroid calf serum depleted of L-T3 and L-T4 by this procedure yields serum which, when used as a medium supplement, results in biological responses identical to those obtained with media supplemented with thyroidectomized calf serum. In addition, resin treatment does not alter the growth-promoting properties of the serum if the thyroid hormone concentration is restored. This procedure should be useful in preparing thyroid hormone-depleted serum for cell culture studies in situations where thyroidectomy is not feasible or would require surgical procedures on a large number of small animals.