The association between infection with HSV and the subsequent development of erythema multiforme is well established, although the role that the virus plays in the pathogenesis of this disorder is not known. HSV DNA has been detected in cutaneous lesions of herpes-associated erythema multiforme (HAEM), and it has been suggested that the tissue damage seen in these lesions is virus-specific. In the current, prospective study, we examined biopsies of lesional, non-involved, and previously involved but healed skin, in addition to specimens of peripheral blood, from patients with HAEM, for HSV DNA by using the polymerase chain reaction. HSV DNA was detected in lesional skin of 10 of 11 patients compared to 2 of 11 non-involved skin biopsies obtained at the same time. HSV was present in 4 of 6 blood specimens obtained during the acute episode. Five patients returned 3 months after the acute episode resolved for biopsies of previously involved skin. HSV was detected in 4 of these 5 biopsies. Thus, the presence of HSV DNA in the skin of patients with HAEM appears to be predominantly in areas of clinical involvement; the virus remains in those cutaneous sites for up to 3 months without evidence of clinical disease; and HSV DNA may be detected in the peripheral blood cells during acute HAEM. Based on these findings, we suggest that the virus plays a role in lesion development, that the skin may function as a site of viral persistence, and that hematogenous spread of viral DNA may be an important factor in the development of HAEM.