The use of aminolevulinic acid (ALA), a precursor of protoporphyrin IX (PPIX), is a new concept in PDT. The goal of our study was to compare two different incubation times of ALA in two cell lines and to evaluate phototoxic potential in correlation with extraction data of PP IX. For PDT a concentration of 1, 5, and 10 mM of ALA for Waf bladder cancer cells and 0.1, 1, and 1.5 mM for Mefa fibroblasts was used. The incubation time was 3.5 or 5.5 h. Irradiation was performed with an argon-dye laser emitting light at a wavelength of 630 nm. After irradiation, cells were incubated for two days and compared with control cells. For extraction of PP IX, cells were incubated with ALA at the same concentration as for PDT experiments for 3.5, 5.5, and 7.5 h. In both cell lines the survival rate was decreased after an incubation time of 5.5 h as compared to 3.5 h independent of the ALA-concentration or the fluence. Light and ALA alone had no effects on cells. The extraction data showed a good correlation with these results. In both cell lines the highest fluorescence of PP IX was measured after an incubation time of 5.5 h.