Significant amounts of protoporphyrin IX (PpIX) are formed after 6 min of topical application of 5-aminolevulinic acid (ALA) and its hexylester derivative, whereas PpIX is formed after 10 min of topical application of ALA-methylester derivative in normal mouse skin at 37°C. Lowering the skin temperature to 28–32°C by the administration of the anesthetic Hypnorm–Dormicum reduces the PpIX fluorescence by a factor of 2–3. Practically no PpIX was formed as long as the skin temperature was kept at 12–18°C. At around 30°C PpIX fluorescence appears later after application of ALA-ester derivatives (14–20 min) than after application of ALA (8 min), indicating differences in their bioavailability (delayed penetration through the stratum corneum, cellular uptake, conversion to ALA, PpIX production) in mouse skin in vivo. The difference in lag time in the PpIX formation after application of ALA and ALA-esters may be partly related to deesterification of the ALA-ester molecules. The temperature dependence of PpIX production may be used for improvement of photodynamic therapy with ALA and ALA-ester derivatives, where accumulation of PpIX can be selectively enhanced by increasing the temperature of the target tissue.