HeLa cells grown in chemically defined medium lacking glucocorticoids synthesize metallothioneins, low molecular-weight heavy-metal binding proteins. Dexamethasone and hydrocortisone increase the rate of metallothionein synthesis five- to ten-fold. Maximal induction is achieved with 10(-8)M dexamethasone and 10 (-7)M hydrocortisone. Half-maximal induction is achieved at 5 X 10(-9)M dexamethasone and 5 X 10(-8)M hydrocortisone. Although carried for many generations in the absence of any glucocorticoids, HeLa cells (clone S) contain 25,000 specific 3H-dexamethasone receptors that translocate into the nucleus after one hour of incubation. 3H-dexamethasone binds to a single class of receptors with an apparent Kd = 18.8 nM. A variety of steroids can be classified into three classes, based on their effect on metallothionein synthesis: (a) full agonists (optimal inducers), (b) intermediate effectors which have either partial agonist or antagonist activities, and (c) inactive steroids. There is a correlation between the effects on metallothionein synthesis of different steroids and their ability to compete with 3H-dexamethasone binding. We conclude that metallothionein is induced in HeLa cells by a glucocorticoid receptor mediated mechanism.