Fluorescence microscopy is an invaluable technique for investigating structural and biochemical changes in cells and tissues. While it is preferable to study these changes in living specimens, such studies are often compromised by the destructive properties of light which can cause cellular damage either directly (photoablation) or indirectly by generating toxic by-products (phototoxicity). To minimize these problems, new methods of illuminating cells are being developed. In particular, ultrafast infrared lasers have been employed to excite fluorophores at one-half and one- third the wavelength of the laser by a process called multiphoton excitation. This process limits excitation to a small volume of indicator which, together with fast scanning of the sample, may reduce photodamage. One source of photodamage is light-induced stimulation of H2O2 in cells. In this report, we tested whether scanning with an ultrafast Ti:sapphire laser could stimulate H2O2 production in cultured human and monkey cells measured with the fluorescent indicator dichlorodihydrofluorescein. We demonstrate that illumination at 800 - 900 nm induced H2O2 production in cells when laser power was increased above 10 mW (at the specimen plane). The frequency of scanning (duty cycle) also influenced H2O2 production indicating that a trade-off between power and exposure time may be an appropriate way to control this type of toxicity. Alternatively, high power and increased exposure time could provide an effective means for controlling H2O2 production and subsequent damage to cellular structures.© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.