Photodynamic therapy of cancer is a promising treatment based on the tumor-specific accumulation of photosensitizers followed by irradiation with visible light which induces tumor cell death. The effect of different preincubation times on the photosensitization efficiency of the phthalocyanines AlPc and AlPcS4 was investigated in lymphoblastoid CCRF-CEM cells under conditions that allow maximal uptake of the sensitizers. First, the time course for the uptake of AlPcS4 and AlPc by CCRF-CEM cells and by the pheochromocytoma PC12 cells was compared. The uptake of AlPcS4 by CCRF-CEM cells was not significantly different after 6 h or 24 h incubation, but the photosensitization efficiency of the phthalocyanine was much higher when a 24 h preincubation period was used, with a fluence rate of 5 mW/cm2. However, for a fluence rate of 10 mW/cm2, the photosensitization efficiency of AlPcS4 was almost completely independent of the preincubation time (6 h vs. 24 h) with the phthalocyanine. When the cells were preincubated with 1 micromol/L AlPc for 10 min or 6 h, which allows the same accumulation of sensitizer by the cells, no significant effect of the incubation time on the photodynamic inactivation of CCRF-CEM cells was observed, with fluence rates of 5 mW/cm2 or 10 mW/cm2, for different light doses. Confocal fluorescence microscopy studies did not reveal differences in the localization of the phthalocyanines after maximal uptake was reached. The results show that the preincubation time with AlPcS4, after the maximal uptake is reached, affects cell growth to an extent depending on the fluence rate used, and this effect was not due to a major redistribution of the sensitizer during incubation. However, this was not observed when AlPc was used.