Other studies have shown that the immunophenotype of Reed-Sternberg and Hodgkin's (RS-H) cells in Hodgkin's disease commonly changes over time, as shown by examination of multiple biopsy specimens obtained from an individual patient. In this study we analyzed 96 sequential biopsy specimens (>1 month apart) obtained from 44 patients (nodular sclerosis, 34 specimens; mixed cellularity, 5; lymphocyte depletion, 1; unclassified, 4) using fixed, paraffin-embedded sections; heat-induced epitope retrieval (HIER); a panel of antibodies specific for the CD3, CD15, CD20, CD30, CD43, CD45/45RB, and CD79a antigens and Epstein-Barr virus latent-membrane protein; and a streptavidin-biotin method. In selected cases in which immunophenotypic changes occurred, studies were repeated using enzyme predigestion instead of HIER. There was no change in the immunophenotype of the RS-H cells in 36 (82%) of 44 patients. In 8 patients (18%), the immunophenotype of the RS-H cells varied in expression of one or two antigens. The antigens that varied were as follows: CD30, 3 patients; CD15, 3 patients; CD20, 1 patient; and CD15 and CD30, 1 patient. We conclude that the immunophenotype of RS-H cells in Hodgkin's disease is relatively stable over time and that CD15 and CD30 are the most common antigens that change. The frequency of immunophenotypic changes, 18%, is substantially lower than that reported previously. One likely explanation for this discrepancy is that we used HIER, a relatively recent innovation in diagnostic immunohistochemistry that has been shown to reduce artifacts attributable to inconsistent fixation and processing. The significance of immunophenotypic variation in eight cases (18%) is uncertain. This phenomenon may represent true biologic changes in RS-H cells. Alternatively, these changes may be attributable to artifacts secondary to inconsistent fixation or processing that HIER cannot overcome.