Hypericin is a natural photo-active pigment produced by plants of the genus Hypericum. The compound exhibits a potent photocytotoxic activity in vitro and in vivo. Using HeLa cells we further investigated whether the photocytotoxic in vitro effect of hypericin is influenced by the cell density. It was demonstrated that hypericin-induced photocytotoxicity in HeLa cells depends significantly on the amount of cells in culture, as low cell density cultures were more responsive to photodynamic therapy (PDT) than confluent or hyperconfluent cell cultures. This confluence dependent resistance (CDR) can be explained in terms of a decrease in hypericin cellular uptake. The phenomenon is not caused by the depletion of hypericin from the medium by high-density cell cultures since the extra-cellular availability of the drug is not altered by the increase in cell density and does not appear to be a limiting factor. Importantly, since confluent or hyperconfluent cell cultures can better mimic the high cell density of the solid tumour, CDR should be taken into consideration whenever resistance of solid tumours to PDT with hypericin is observed.