BACKGROUND AND OBJECTIVE Graft-versus-host disease (GVHD) presents an important complication of allogeneic bone marrow transplantation. A method to predict GVHD might be the analysis of cytotoxic T lymphocyte precursors, but the technique requires the use of radioactive elements not suitable in all laboratories. DESIGN AND METHODS Serine esterase (SE) activity was studied by a cytochemical method in donor-recipient mixed lymphocytes cultures (MLC). Twelve patients, affected by acute or chronic leukemia, and 20 donors were studied. MLC incubated with and without growth factors (IGF-1 or IL-2), were analyzed. The relationship between positivity of MLC and GVHD in transplanted patients was evaluated. RESULTS The data obtained showed that the percentage of SE positive cells was higher in MLC compared to negative control MLC. The highest percentage of positive cells was found in the MLC obtained from unrelated subjects. INTERPRETATION AND CONCLUSIONS These results show that serine protease expression in MLC may be a predictive marker of GVHD and could be used as an additional early test associated with cytotoxicity.