Although singlet oxygen ((1)O(2)) has long been proposed as the primary reactive oxygen species in photodynamic therapy (PDT), it has only recently been possible to detect it in biological systems by its luminescence at 1270 nm. Having previously demonstrated this in vitro and in vivo, we showed that cell survival was strongly correlated to the (1)O(2) luminescence in cell suspensions over a wide range of treatment parameters. Here, we extend this to test the hypothesis that the photobiological response in vivo is also correlated with (1)O(2) generation, independent of individual treatment parameters. The normal skin of SKH1-HR hairless mice was sensitised with 20% amino-levulinic acid-induced protoporophyrin IX and exposed to 5, 11, 22 or 50 J cm(-2) of pulsed 523 nm light at 50 mW cm(-2), or to 50 J cm(-2) at 15 or 150 mW cm(-2). (1)O(2) luminescence was measured during treatment and the photodynamic response of the skin was scored daily for 2 weeks after treatment. As observed by other authors, a strong irradiance dependence of the PDT effect was observed. However, in all cases the responses increased with the (1)O(2) luminescence, independent of the irradiance, demonstrating for the first time in vivo an unequivocal mechanistic link between (1)O(2) generation and photobiological response.