The major histocompatibility complex class I (MHC-I) has recently been shown not only to present antigens to the immune system but also to mediate transmembrane signaling, resulting in activation, inactivation, or apoptosis. Such signaling has been observed in both normal and malignant cells of the B and T cell lineage. Cross-linking of MHC-I on the pre-B-acute-lymphocytic cell line KM-3 induces an apoptotic process, which becomes evident after approximately 12 h. In order to better understand the mechanisms regulating this apoptotic process, we have investigated both gene expression and the effect of cross-linking on certain intracellular events. Differential display PCR was used to isolate two gene fragments whose level of expression was associated with the induction of apoptosis as they were downregulated in KM-3 cells following MHC-I cross-linking. These genes encode novel molecules whose function remains to be elucidated. It was further demonstrated that the apoptotic process was not accompanied by changes in [Ca2+]i, the level of activation of NF-kappaB, or changes in protein kinase C activity and that the initiation of apoptosis could be prevented by phorbol ester treatment. It is thus suggested that multiple, fine-tuned molecular events determine the outcome of cross-linking of MHC-I in this pre-B-lymphocytic cell line.