The aim of our study was to find out if Photofrin II, a cytotoxic drug used routinely in photodynamic therapy (PDT), can induce immune responses in vitro, and to compare its effects with those of the protoporphyrin 9, hemin, which also has antitumor properties. We tested the effect of these porphyrins on lymphocyte proliferation and secretion of interleukin-2, interleukin-3, tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma), by human or murine mononuclear cells (MNC) without an activating light. Both the Photofrin II- and hemin-treated cells showed a significant increase in cytokine secretion in the presence of suboptimal concentrations of mitogen. Moreover, Photofrin II and hemin significantly increased production of TNFalpha and IFNgamma even in the absence of mitogen. The cellular binding sites of Photofrin II and hemin to MNC were localized by electromicroscopy or fluorescence. Combined stimulation of cells by mitogens and porphyrins maintained optimal vital ionic balance of potassium, sodium and chlorine in the lymphocytes. In the cells thus treated there was a significant increase in intracellular calcium, a vital second messenger for lymphokine secretion. We demonstrate that the effect of Photofrin II on the immune system involves enhanced cytokine secretion which may account for the subsequent tumor eradication by PDT.