Light fractionation with dark periods of the order of hours has been shown to considerably increase the efficacy of 5-aminolevulinic acid-photodynamic therapy (ALA-PDT). Recent investigations have suggested that this increase may be due to the resynthesis of protoporphyrin IX (PpIX) during the dark period following the first illumination that is then utilized in the second light fraction. We have investigated the kinetics of PpIX fluorescence and PDT-induced damage during PDT in the normal skin of the SKH1 HR hairless mouse. A single illumination (514 nm), with light fluences of 5, 10 and 50 J cm⁻² was performed 4 h after the application of 20% ALA, to determine the effect of PDT on the synthesis of PpIX. Results show that the kinetics of PpIX fluorescence after illumination are dependent on the fluence delivered; the resynthesis of PpIX is progressively inhibited following fluences above 10 J cm⁻². In order to determine the influence of the PpIX fluorescence intensity at the time of the second illumination on the visual skin damage, 5 95 and 50 50 J cm⁻² (when significantly less PpIX fluorescence is present before the second illumination), were delivered with a dark interval of 2 h between light fractions. Each scheme was compared to illumination with 100 J cm⁻² in a single fraction delivered 4 or 6 h after the application of ALA. As we have shown previously greater skin damage results when an equal light fluence is delivered in two fractions. However, significantly more damage results when 5 J cm⁻² is delivered in the first light fraction. Also, delivering 5 J cm⁻² at 5 mW cm⁻² 95 J cm⁻² at 50 mW cm⁻² results in a reduction in visual skin damage from that obtained with 5 95 J cm⁻² at 50 mW cm⁻². A similar reduction in damage is observed if 5 45 J cm⁻² are delivered at 50 mW cm⁻². PpIX photoproducts are formed during illumination and subsequently photobleached. PpIX photoproducts do not dissipate in the 2 h dark interval between illuminations.