ICR 27 is a mutant derived from the glucocorticoid-sensitive human leukemic cell line CEM C7 that has been characterized as glucocorticoid receptor negative based on its ability to specifically bind [3H]dexamethasone ([3H]DEX). We used the pyrazolosteroid [3H]cortivazol ([3H]CVZ) to determine whether ICR 27 cells actually contain glucocorticoid receptors and, if so, whether these receptors can mediate physiological effects. Scatchard analysis of the binding of [3H]CVZ to cytosol from ICR 27 cells was consistent with a single class of receptors of uniform affinity (0.7 nM). Cytosolic [3H]CVZ complexes had a sedimentation coefficient of 4.6S on linear sucrose gradients and eluted from DEAE-cellulose columns at a potassium phosphate concentration of 250 mM. CVZ also competed with [3H]DEX mesylate for binding to a 96,000 mol wt protein. Incubation of ICR 27 cells with CVZ caused 50% growth inhibition and 50% maximal induction of glutamine synthetase activity at concentrations of 20 and 35 nM, respectively. Elution profiles of [3H]CVZ complexes from DEAE-cellulose columns showed that complexes formed upon thermal activation were relatively unstable, and little or no increase in binding of [3H]CVZ-receptor complexes to DNA-cellulose was observed. Thus, [3H]CVZ identifies functional glucocorticoid receptors in a cell line previously described as DEX resistant. Although the binding of [3H]CVZ to activated receptors in vitro appears unstable, high concentrations of CVZ may facilitate stabilization of activated complexes that can mediate both anabolic and catabolic effects.