The in vivo wavelength dependence of hematoporphyrin derivative (HpD) photodynamic treatment (PDT) has been studied. Ears of 136 rats were treated at six red and four blue-green laser wavelengths (615-635, 488-514.5 nm). Hematoporphyrin derivative was administered intraperitoneally (15 mg/kg) and 24 h later both ears were irradiated, at different wavelengths, for t = 6.5, 10 or 15 min at 60 mW/cm2. Four parameters (thickness, average erythema, eschar and loss of tissue) were quantified and a combined score (CS) of effects was established statistically. The maximum combined score during follow-up was taken as a measure for the biological effect. The light distribution in rat ears during irradiation with red and blue-green light was estimated from in vivo measurements and the transport theory. Statistical analysis of the combined score data yielded values for the relative biological effectiveness (RBE). Relative biological effectiveness maxima occurred at 501.7 and 625 nm. Analyzing erythema and loss of tissue separately yielded maxima at the same wavelengths. Quantitative agreement between the latter two sets of relative biological effectiveness values was obtained only when they were referred to the actual light energy fluence in tissue, rather than to the incident fluence. These relative biological effectiveness values are about 2.3 at 501.7 nm and 1.35 at 625 nm, taking relative biological effectiveness = 1 at 630 nm.