Photodynamic therapy (PDT) with topical d-aminolevulinic acid (ALA) has become a therapeutic option of growing interest for superficial non-melanoma precancerous and malignant lesions. After application of ALA, in situ conversion to endogenous porphyrins is accomplished in a gradual manner. Therefore, the determination of fluorescence kinetics and spatial distribution in vivo versus time is a crucial point for the success of ALA-PDT. Seventeen basal cell carcinomas (BCC) and 20 actinic keratoses (AK) were enrolled in this study. In 5 BCC and 4 AK, in vivo fluorescence kinetics were performed over 24 hrs and for the remaining lesions between 2 and 7 hrs after ALA application. In vivo spatial and quantitative detection of the fluorescence intensity versus time showed considerable variations among tumors of the same type, so light irradiation was performed according to patient individualities. Both BCC and AK showed maximal median fluorescence intensity at 4-6.5 hrs post-application. In the present study, a high cure rate was proven after topical ALA-PDT (70.6% in BCC and 85% in AK). The results of fluorescence studies suggest that optimum irradiation time for BCC is approximately 3.5-5 hrs and for AK 5 hrs after ALA application, when relative maximal fluorescence intensity in correlation with fluorescence selectivity on the lesion, is obtained.