The response of human glioma spheroids to 5-aminolevulinic acid (ALA)–mediated photodynamic therapy (PDT) is investigated. A two-photon fluorescence microscopy technique is used to show that human glioma cells readily convert ALA to protoporphyrin IX throughout the entire spheroid volume. The central finding of this study is that the response of human glioma spheroids to ALA-mediated PDT depends not only on the total fluence, but also on the rate at which the fluence is delivered. At low fluences (≤50 J cm⁻²), lower fluence rates are more effective. At a fluence of 50 J cm⁻², near-total spheroid kill is observed at fluence rates of as low as 10 mW cm⁻². The fluence rate effect is not as pronounced at higher fluences (>50 J cm⁻²), where a favorable response is observed throughout the range of fluence rates investigated. The clinical implications of these findings are discussed.