A fractionated illumination scheme in which a cumulative fluence of 100 J cm⁻² is delivered in two equal light fractions separated by a dark interval of 2 h has been shown to considerably increase the efficacy of 5-aminolevulinic acid (ALA)–photodynamic therapy (PDT). The efficacy of such a scheme is further increased if the fluence of the first light fraction is reduced to 5 J cm⁻². We have investigated the relationship between the PDT response and the kinetics of protoporphyrin IX (PpIX) fluorescence in the SKH1 HR hairless mouse for first fraction fluences below 5 J cm⁻² delivered 4 h after the application of ALA and 10 J cm⁻² delivered 2 h after the application of ALA. Illumination is performed using 514 nm at a fluence rate of 50 mW cm⁻². Reducing the fluence of the first fraction to 2.5 J cm⁻² does not result in significantly different visual skin damage. The PDT response, however, is significantly reduced if the fluence is lowered to 1 J cm⁻², but this illumination scheme (1 99 J cm⁻²) remains significantly more effective than a single illumination of 100 J cm⁻². A first light fraction of 10 J cm⁻² can be delivered 2 h earlier, 2 h after the application of ALA, without significant reduction in the PDT response compared with 5 95 J cm⁻² delivered 4 and 6 h after the application of ALA. The kinetics of PpIX fluorescence are consistent with those reported previously by us and do not explain the significant increase in PDT response with a two-fold illumination scheme. Histological sections of the illuminated volume showed a trend toward increasing extent and depth of necrosis for the two-fold illumination scheme in which the first light fraction is 5 J cm⁻², compared with a single illumination scheme.