Ultraviolet (UV) radiation is one of the major environmental harmful factors that affect human skin. Numerous evidences have indicated that UVB radiation was a potent inducer of reactive oxygen species (ROS) production, which can lead to the oxidative generated modification of various cellular molecules. In present studies, the 3T3 fibroblasts were exposed to 5 similar to 50 mJ/cm(2) of UVB radiation without the overlay PBS for 10, 20, 30, and 40 minutes, respectively. The results indicate that the fluorescence intensity of ROS in mouse 3T3 fibroblasts is increased with the increasing time of UVB radiation exposure. However, the fluorescence intensity of mitochondria in mouse 3T3 fibroblasts is decreased with the increasing time of UVB radiation exposure. Moreover, the fluorescence intensity of ROS in mouse 3T3 fibroblasts exposed to 10, 20, 30, and 40 minutes was all very significantly higher than the control group, while the fluorescence intensity of mitochondria in mouse 3T3 fibroblasts exposed to 10, 20, 30, and 40 minutes was all very significantly lower than the control group. Taken together, the effects of UVB irradiation on the fluorescence intensity of ROS and mitochondria in mouse 3T3 fibroblasts can be analyzed easily and conveniently with confocal microscope and confocal software.