The prevailing view regarding the mechanism of steroid cataract formation holds that glucocorticoids are covalently bound to lens proteins resulting in destabilization of the protein structure allowing further modification (i.e. oxidation) leading to cataract. Alternative hypotheses (e.g. that cataracts result from glucocorticoid receptor mediated effects) have been difficult to test since protein binding does in fact occur for many cataractogenic steroids. A glucocorticoid lacking the typical glucocorticoid hydroxy group at C21 (fluorometholone, FML), other steroids which can bind to proteins but lack glucocorticoid activity, and a glucocorticoid antagonist (RU486) have been utilized to discriminate between these two hypotheses. Purified bovine beta-crystallin incubated with three different 3H-steroids, dexamethasone (Dex), aldosterone or progesterone demonstrated that the C-21 hydroxyl group is not essential for steroid binding. Progesterone (with no C-21 OH) bound to the greatest extent. Pretreatment of the protein with aspirin to acetylate the free protein amino groups blocked this binding, demonstrating the probability of a Schiff base mechanism. Lens culture studies with the same three radiolabeled steroids demonstrated much the same result. Rat lenses cultured for 48 hr-11 days, demonstrated that loss of GSH is an early and significant effect of several glucocorticoids (Dex, prednisolone and FML) but is not seen with other non-glucocorticoid steroids. However, none of the steroids tested consistently produced lenticular opacification (i.e. cataracts) in this in vitro system, nor did they alter rubidium transport. We suggest that a mechanism other than covalent binding of steroids to lens proteins is responsible for glucocorticoid induced cataracts because: (1) non-glucocorticoids were demonstrated to bind lens proteins as well or better than the glucocorticoid Dex and (2) only glucocorticoids, and not other steroids, lowered lens reduced glutathione content which has been demonstrated to be associated with other forms of cataract.