The diagnosis of extramedullary plasmacytomas (EMP) is usually easy to conform with immunohistochemical stains for kappa and lambda. In some cases, however, immunostains are problematic. In addition, prognostic features are not well described nor is it known whether EMP are associated with the Epstein-Barr virus. Therefore, 23 EMP of the head and neck (from 20 patients) were studied to (1) compare a non-isotopic paraffin section in situ hybridization technique for kappa and lambda mRNA with standard immunohistochemical techniques for assessing light chain expression, (2) compare the histologic grade to the proliferative fraction using an antibody for the proliferating cell nuclear antigen, and (3) determine the frequency of Epstein-Barr virus (EBV) association using probes for the EBV DNA (EBV NOT 1) and RNA (EBER-1). Light chain class restriction was demonstrated in 22/23 biopsies by in situ hybridization and in 21/23 biopsies by standard immunohistochemical techniques. Five of the six biopsies of well-differentiated EMP had proliferating cell nuclear antigen scores of 0 to 10% positive cells and one had 11 to 25% positive cells compared with greater than 75% in the one poorly differentiated EMP. Of 15 moderately differentiated EMP, 10 had proliferating cell nuclear antigen scores of 11 to 75%, and 5 had scores of greater than 75%. EBV DNA was detected in 1/23 biopsies and EBV RNA in 4/23 biopsies (3 patients). Thus, non-isotopic in situ hybridization is a useful technique to document clonality of plasma cells in routinely fixed, paraffin-embedded sections. Unlike routine immunohistochemistry, in situ hybridization avoids the problem of detecting stromal or nonspecific uptake of immunoglobulin.(ABSTRACT TRUNCATED AT 250 WORDS)