Selection procedures in bone marrow transplantation (BMT) would benefit from the development of easy-to-perform cellular assays with high discriminative power. We tested a cytokine-based mixed lymphocyte culture (MLC) and compared its outcome to the routinely used MLC, helper T-lymphocyte precursor (HTLp)-f and cytotoxic T-lymphocyte precursor (CTLp)-f assays. Interferon (IFN)gamma was selected as a marker cytokine for (deleterious) T-helper 1 like responses and 36 (potential) BMT donor-recipient pairs were analysed. The IFNgamma-MLC appeared sensitive to HLA class II (subtype) differences, but not to isolated class I differences, or to mismatches other than HLA (identical siblings). The test enabled a distinction between combinations with positive MLC (proliferation) and HTLp-f, exemplified by the fact that although high IFNgamma levels were observed in the class II mismatched group, certain DRB3, DQB1-subtype and DRB1-subtype mismatches did not give rise to IFNgamma production. This might be of relevance for the detection of so-called permissible mismatches. With regard to prediction of acute graft-versus-host disease (aGVHD) in unrelated BMT, the data indicated that high levels of IFNgamma coincided with severe aGVHD, whereas low levels were largely associated with grades 0-I. However, in the case of isolated class I mismatches the test had no predictive value. The cell-saving IFNgamma-MLC provides an alternative for the assays currently in use, but should be employed along with an assay that is sensitive to class I differences to correct for false negatives. Consequently, a combination of IFNgamma-MLC and CTLp-f assays seems most promising for donor selection, other than identical siblings.