The high toxicity of reactive oxygen species (ROS) suggested a possible role in the pathogenicity of human pathogenic fungi. We previously reported a chemiluminescence method for measuring ROS generation in Candida albicans. In the present study, we attempted to visualize the ROS, superoxide anion radical (O-2(-)), generated by paraquat (PQ)-stimulated C albicans using methyl-Cypridina-luciferin analog (MCLA) as a chemiluminescence probe. Colonies of a wild-type C albicans parent strain and its respiration-deficient mutant grown on agar plates were overlaid with a mixture of PQ and MCLA solutions. MCLA-dependent light emission from the colonies was recorded with a Hamamatsu ultralow-light-imaging apparatus with a CCD camera in a light-tight box. In the wild-type strain, marginal regions of growing colonies were strongly illuminated. The light emission from the colonies was extinguished by superoxide dismutase (SOD), proving that the light emission was strictly clue to the superoxide anion. However, colonies of the respiration-deficient mutant poorly generated superoxide. Chemiluminecence measurements by a luminometer showed vigorous superoxide generation by the exponential phase cells of the parent strain but weak generation by the stationary phase cells. In the mutant, superoxide generation was weak compared with the parent strain. These results indicate that expansion of the colonies was due to the actively respiring cells located in the marginal regions. To our knowledge, the present report is the first chemiluminescent visualization of ROS including superoxide generated by C albicans.