The mechanism of pemphigus acantholysis has been studied with an in vitro system. Freshly prepared human skin epidermal cells were incubated in F-10 medium which contained the immunoglobulin G fraction from either pemphigus serum or normal human serum. During 18-h incubation periods, the pemphigus antibody became bound to the surface of the epidermal cells, caused the destruction of 75% of the viable cells as compared to only 14% in the normal immunoglobulin G controls (trypan blue exclusion), prevented the accumulation of newly synthesized proteins by nearly 60% as determined by radioactive tracer studies, and caused a dramatic shift in distribution of the newly synthesized proteins from an insoluble cell-associated fraction to an extracellular soluble fraction. These effects on the accumulation and partitioning of newly synthesized proteins were antibody concentration-dependent. Kinetic studies showed that at a fixed pemphigus antibody concentration the inhibition of protein accumulation preceded solubilization by about 1 h, at which time rapid solubilization of up to 70% of the insoluble cellular material occurred. Several lines of evidence suggested that this phenomenon was caused by enzymatic activity. Epidermal extracts solubilized a prepared substrate of radioactivity labeled insoluble epidermal cell material. This activity was heat labile and pH dependent, with pH optima ranging from 4.5 to 6.5. Enzymes with pH optima between 6 and 6.5 were recovered in the culture medium after a 2-day incubation of pure, intact epidermis with the pemphigus antibody. We proposed the following hypothesis to account for pemphigus acantholysis. The pemphigus antibody reacts with the epidermal cell surface and produces such a severe disturbance that the integrity of the cell surface is lost. As a result of these primary perturbations, the cell is killed and during the process, responds by release or activiation of soluble hydrolytic enzymes. This autolytic process results in the characteristic acantholysis of pemphigus.