Photodynamic therapy (PDT) can induce direct tumor cell destruction, indirect tumor cell inactivation due to vascular occlusion as well as immune response. Evidence suggests that the PDT-induced cell death is dependent on both PDT protocol-related as well as microenvironmental factors, and its mode is also decisive for the type of immune response. This suggests potential interrelationship among PDT-induced tumor cell destruction, immune response and microenvironmental factors. In the present study we analyzed the effect of a microenvironmental factor - extracellular matrix on the cellular response to photodynamic treatment in vitro. By using conventional proliferation and modified cell survival assays as well as fluorescence imaging, we compared efficacy of aminolevulinic acid (ALA)-PDT to inactivate three esophageal cell lines in two- and three-dimensional formats. Modified cell colony assay indicated comparable PDT doses leading to death of both Kyse 450 and Het-1A cells on plastic, whereas Kyse 70 cells were only partially responsive. In 3D collagen matrices, we were able to induce only death of Kyse 450 cells by ALA-PDT, if analyzed 24h after treatment. Consistently, only Kyse 450 cells were able to produce detectable amounts of PpIX after incubation of their 3D collagen cultures with ALA. Our results demonstrate that cellular response to ALA-PDT is cell-type dependent both in two- and three-dimensional formats and indicate that the extracellular matrix might modify it.