The objective of this study was to evaluate the efficacy of immunohistochemical (IHC) staining for diagnosis of persistent bovine viral diarrhea virus (BVDV) infection using formalin-fixed, paraffin-embedded skin biopsy specimens. Skin from 41 of 42 calves shown to be persistently infected (PI) with BVDV by repeated virus isolation more than 3 weeks apart were immunohistochemically positive for BVDV antigen. Positive IHC staining was most pronounced in the keratinocytes and in hair follicle epithelium, hair matrix cells of the hair bulb, and the dermal papilla. All of the skin sections from 10 calves experimentally infected postnatally with BVDV (10(5) median tissue culture infective doses [TCID50]) and biopsied on days 0, 5, 7, and 9 postinfection were negative for viral antigen. Ten calves from a second group experimentally infected with a higher dose of BVDV (10(8) TCID50) were biopsied when viremic between 10 and 14 days postinfection and 4 calves exhibited positive IHC staining for BVDV; however, staining in these skin biopsies was confined to small foci in the nonfollicular epidermis and follicular ostia. This staining was distinct from that observed in skin obtained from PI cattle. Skin biopsy represents an effective method for identifying animals PI with BVDV.