The glucocorticoid receptors were measured and characterized in the transplantable B16 murine melanoma using [3H]-dexamethasone by a charcoal adsorption technique. In the tumor cytosols assayed, the levels of receptors ranged from 44 to 200 fmol/mg protein, and the corresponding Kd's ranged from 2 to 43 nM. Sucrose density gradient analysis showed a peak sedimenting at 7.1S under low-ionic-strength buffer which was completely eliminated with a 100-fold molar excess of unlabeled triamcinolone acetonide in the incubation mixture. This peak of bound radioactivity shifted to the 4.4S region under high-ionic-strength buffer (0.4 M KCl) conditions. Competition experiments, using [3H]dexamethasone and various unlabeled steroids at a 100-fold molar excess, showed characteristics typical of glucocorticoid receptors seen in other tissues. Administration of various glucocorticoids, e.g., dexamethasone, hydrocortisone acetate, and prednisolone, in different doses and regimens showed a marked and significant inhibition of tumor growth as measured by mean tumor diameter and weight. Although glucocorticoid treatment does not seem to affect the incidence of pulmonary metastases, the number of pulmonary nodules appears to be significantly greater in some groups treated with higher doses of these drugs. In survival experiments, administration of hydrocortisone acetate in various doses and regimens also resulted in a significant increase in the median survival of mice compared to 0.9% NaCl solution-treated controls. These results indicate that the growth inhibition of B16 melanoma by glucocorticoids may be a direct effect mediated by interaction with the glucocorticoid receptor.