Glucocorticosteroids are used to treat patients with pemphigus, but the mechanism of action is unknown. We studied the effect of methylprednisolone on acantholysis induced in vitro by incubation of normal skin with plasma from a patient with pemphigus. Normal human breast skin was maintained in organ cultures for several days in Ham F-10 medium. Plasma from a patient with active pemphigus vulgaris caused suprabasilar epidermal acantholysis when added to this culture system. In control cultures (F-10 medium and fetal bovine serum), no acantholysis occurred. Acantholysis was prevented when breast skin was preincubated for 24 h in a 0.25 mM solution of methylprednisolone in F-10 medium and fetal bovine serum, suggesting that methylprednisolone directly inhibits acantholysis. No suppression of acantholysis occurred when the methylprednisolone was added to the culture system simultaneously with the pemphigus plasma, suggesting a time requirement for alteration of cellular events. The inhibition of acantholysis was not caused by cell death since methylprednisolone did not alter keratinocyte viability as determined by exclusion of trypan blue dye when keratinocytes were exposed to pemphigus plasma. Similarly, the inhibition of acantholysis was not due to dissolution, alteration, or coating of pemphigus antigen on epidermal cells, since the intercellular antibodies in the plasma bound as well to methylprednisolone-treated epidermis as to untreated epidermis.