An alloreactive reaction similar to that occurring during GvHD can be generated in a mixed lymphocyte culture. The presence of both stimulator and responder cells in these cultures makes the identification and enumeration of alloreactive cells difficult and unreliable. We describe the use of PBMC sonicates as an alternative to the standard MLC method to stimulate an allogeneic reaction. Using combinations of autologous or allogeneic PBMC sonicates, we showed that the lymphocyte proliferative response to cell sonicates was comparable to the response using irradiated cells. The proliferative response was concentration dependent and reached maximum levels at day 6. Both irradiated cells and PBMC sonicates induced significantly lower responses when the stimulating cells were partially HLA-DR matched rather than completely mismatched. Alloreactive T cells stimulated with sonicates were enumerated by the flow cytometric detection of CD69 or CD25. In HLA-mismatched cultures, approximately 7% of CD3+ T cells were CD69+ or CD25+, suggesting alloreactivity. Although there was a significant correlation between the expression of these activation markers and lymphocyte proliferative responses, significant individual variations in the results of these two assays were observed. The results in this study demonstrate the potential of using PBMC sonicates instead of irradiated lymphocytes for the study and identification of alloreactive cells at the cellular and molecular level.