Summary A treatment regimen that takes advantage of the induction of intracellular porphyrins such as protoporphyrin IX (PPIX) by exposure to exogenous 5-amino-laevulinic acid (ALA) followed by localized exposure to visible light represents a promising new approach to photodynamic therapy (PDT). Acting upon the suggestion that the effectiveness of ALA-dependent PDT may depend upon the state of cellular differentiation, we investigated the effect of terminal differentiation upon ALA-induced synthesis of and the subsequent phototoxicity attributable to PPIX in primary mouse keratinocytes. Induction of keratinocyte differentiation augmented intracellular PPIX accumulation in cells treated with ALA. These elevated PPIX levels resulted in an enhanced lethal photodynamic sensitization of differentiated cells. The differentiationdependent increase in cellular PPIX levels resulted from several factors including: (a) increased ALA uptake, (b) enhanced PPIX production and (c) decreased PPIX export into the culture media. Simultaneously, steady-state levels of coproporphyrinogen oxidase mRNA increased but aminolaevulinic acid dehydratase mRNA levels remained unchanged. From experiments using 12-o-tetradecanoylphorbol-13-acetate, transforming growth factor beta 1 and calcimycin we demonstrated that the increase in PPIX concentration in terminally differentiating keratinocytes is calcium-and differentiation specific. Stimulation of the haem synthetic capacity is seen in primary keratinocytes, but not in PAM 212 cells that fail to undergo differentiation. Interestingly, increased PPIX formation and elevated coproporphyrinogen oxidase mRNA levels are not limited to differentiating keratinocytes; these were also elevated in the C2C12 myoblast and the PC12 adrenal cell lines upon induction of differentiation. Overall, the therapeutic implications of these results are that the effectiveness of ALA-dependent PDT depends on the differentiation status of the cell and that this may enable selective targeting of several tissue types.Keywords: photodynamic therapy; 5-aminolaevulinic acid; differentiation; keratinocyte; protoporphyrin IX Photodynamic therapy (PDT) is a treatment strategy consisting of two components: the photosensitizer (PS) and light (Hasan and Parrish, 1996). Both show negligible toxicity by themselves at the doses used for therapeutic applications, but become cytotoxic once combined at the site of desired activity by a variety of photochemical and cellular mechanisms (Henderson and Dougherty, 1992). Current clinical and experimental protocols involve systemic administration of a PS, usually a tetrapyrrole compound, followed by local irradiation with activating light (Fisher et al, 1995), and considerable experience has been acquired with the use of exogenous porphyrins and porphyrin derivatives (Ortel et al, 1996).A more recent approach exploits the indigenous ability of most cells to synthesize porphyrins from their physiological precursor, 5-aminolaevulinic acid (ALA; Batlle, 1993). The synthesis of ALA, the precur...