This paper reports that a new photobleaching compound, 2-(2-chloro-5-propoxycarbonylphenyl)aminomethylidene-5,5-dimethyl-cyclohexane-1,3-dione (RWH-21), stimulates accumulation of 13²-hydroxychlorophyll a in cultured tobacco cells. This was shown based on isolation of 13²-hydroxychlorophyll a from pigment extracts of cultured tobacco cells by diode-array HPLC and subsequent fast atom bombardment mass spectrometry analysis. 13²-Hydroxychlorophyll a rapidly accumulates in tobacco cells both in the light and dark in the presence of RWH-21 (50 μM). Analysis of 13²-hydroxychlorophyll a formation in tobacco cells indicates that 13²-hydroxychlorophyll a is rapidly accumulated within 20 h incubation time both in the dark and light. Although the amount of 13²-hydroxychlorophyll a is continuously increased in the dark, the amount of 13²-hydroxychlorophyll a decreased remarkably in the light after 20 h incubation. Analysis of 13²-hydroxychlorophyll a formation and lipid peroxidation by determination malondialdehyde in tobacco cells suggests that RWH-21-induced 13²-hydroxychlorophyll a has the potential to cause a photodynamic action in cultured tobacco cells.