In order to obtain information about the activation of macrophages (M&s) during photodynamic therapy (PDT), the influence of Photofrin II (Pf II) on the viability of thioglycollate-elicited murine M4.s and the subsequent generation of superoxide anion was studied. Irradiations were performed at an energy density of 5 J cme2, a power density of 150 mW cmm2 and a wavelength of 405 run. Viability of M& was assessed using the acridine orange-ethidium bromide assay. Superoxide anion generation was determined using ferricytochrome c (cyt c) and nitroblue tetrazolium (NBT) reduction. Our results indicate that the MC@ are highly susceptible to PDT as their viability is decreased to approximately 30% by 1 pg ml-' Pf II at the energy density indicated above. Within the tirst 30 min of addition of the photosensitizer, a reducing agent is generated intracellularly by the stimulation of the M&s. An extracellular release of superoxide anion does not occur, as measured by the cyt c assay. Preincubation of the ceils for 1 or 24 h with Pf II and a second challenge with phorbol myristate acetate (PMA) does not enhance the reduction of NBT. Thus, Pf II exerts an immediate effect on the M+s which could be interpreted as a first step for subsequent reactions.