Sera from 20 patients with antiepiligrin cicatricial pemphigoid were studied to define the specific reactivity of their IgG autoantibodies. IgG from all patients bound exclusively to the dermal side of 1 mol/L NaCl split skin and immunoprecipitated laminin 5 (α3β3γ2) from extracts of human keratinocytes (HKs). Immunoblot studies on purified laminin 5 subunits demonstrated that patient IgG bound α3 alone in 16 patients. In two patients, IgG autoantibodies were directed predominantly to the γ2 subunit, yet showed trace reactivity to α3 as well. Sera from two patients did not immunoblot any laminin 5 subunits, their IgG presumably immunoprecipitating laminin 5 via a conformational epitope. Sera from patients with α3 subunit-specific IgG immunoprecipitated all subunits of laminin 5 as well as polypeptides of 190 and 200 kDa from the conditioned media of HKs. Preclearance studies and experiments utilizing affinity-purified patient IgG demonstrated that the latter signified laminin 6 (α3β1γ1) that was bound by cross-reactive α3 subunit-specific patient IgG. Sera from patients with γ2 subunit-specific IgG showed no reactivity to laminin 6, except for faint reactivity provided by low levels of their α3 subunit-specific IgG. Taken together, these findings indicate that antiepiligrin cicatricial pemphigoid signifies an autoimmune response to subunits present in laminin 5.