The ileal Peyer's patch (PP) is the major site of B cell production and is a site of immunoglobulin gene diversification in the sheep. Within the ileal PP follicles there is both intense proliferation and death of B cells. We have previously demonstrated that most, if not all of this death can be attributed to apoptosis. Likewise, ileal PP B cells die rapidly by apoptosis in culture--after 6 h many cells appear pyknotic and about 50% of cellular DNA is fragmented. We now show that the DNA fragmentation and cell death of ileal PP B cells can be almost completely abrogated during the first 12 h of culture by the addition of the phorbol esters, phorbol dibutyrate (PBu2) or phorbol myristate acetate. This inhibition of apoptosis could be sustained for greater than 24 h by the concomitant addition of both PBu2 and the Ca2+ ionophore A23187. However, the rescue of B cells from apoptosis by PBu2, with or without Ca2+ ionophore, was prevented by macromolecular synthesis inhibitors or inhibitors of protein kinase C activation. Furthermore, treatment of cultures with PBu2, with or without Ca2+ ionophore, resulted in an activated B cell phenotype and a three- to fourfold increase in cell proliferation. We conclude that protein kinase C activation in conjunction with an increase in intracellular [Ca2+] can provide the signals necessary to rescue ileal PP B cells from apoptosis, and speculate that these ileal PP B cells are destined to die unless they receive a signal that rescues them from the death pathway.