One of the clinical limitations of the photodynamic therapy (PDT) is the reduced light penetration into biological tissues. Pulsed lasers may present advantages concerning photodynamic response when compared to continuous wave (CW) lasers operating under the same average power conditions. The aim of this study was to investigate PDT-induced response when using femtosecond laser (FSL) and a first-generation photosensitizer (Photogem) to evaluate the induced depth of necrosis. The in vitro photodegradation of the sensitizer was monitored during illumination either with CW or an FSL as an indirect measurement of the PDT response. Healthy liver of Wistar rats was used to evaluate the tissue response. The photosensitizer was endovenously injected and 30 min after, an energy dose of 150 J cm(-2) was delivered to the liver surface. We observed that the photodegradation rate evaluated via fluorescence spectroscopy was higher for the FSL illumination. The FSL-PDT produced a necrosis nearly twice as deep when compared to the CW-PDT. An increase of the tissue temperature during the application was measured and was not higher than 2.5 °C for the CW laser and not higher than 4.5 °C for the pulsed laser. FSL should be considered as an alternative in PDT applications for improving the results in the treatment of bulky tumors where higher light penetration is required.