5-ALA-induced protoporphyrin IX (PPIX) fluorescence kinetics was quantified by fluorescence microscopy in three-dimensional organ co-cultures of human bronchial epithelium, which were infiltrated by four different lung tumour cell lines (EPLC-M31, LCLC-103H, NCI-H125 and NCI-H841). Corresponding fluorescence measurements were performed in monolayer cultures of these tumour cell lines and BEAS-2B cells as a model for normal bronchial epithelium by flow cytometry. Significant differences of fluorescence intensities (FI) between the tumours were detected in organ co-cultures as well as in single cell measurements. Relative FI values in organ co-cultures (FI(EPLC-32M1)>FI(LCLC-H103)>FI(NCI-H125)>FI(NCI-H841)) did not correspond to the measurements in single cells (FI(LCLC-H103)>FI(NCI-H125)>FI(NCI-H841)>FI(EPLC-32M1)). Histology of organ co-cultures revealed different patterns of invasion and tumour cell densities depending on the tumour type. After correction of FI in the co-cultures to tumour cell density the correlation coefficient for fluorescence values between both models increased considerably. Thus, additionally to distinctive features of 5-ALA metabolism, patterns of tumour invasion may be a factor determining 5-ALA-induced fluorescence. Considering these results, a pronounced heterogeneity of 5-ALA-induced fluorescence might be expected in different bronchial tumours in vivo. This could interfere with the diagnostic reliability of 5-ALA-induced fluorescence for early tumour detection.