The number of pigments in single light-harvesting complexes (chlorosomes) were calculated by imaging single chlorosomes in a frozen buffer at cryogenic temperature with a confocal laser fluorescence microscope and pigment extraction. Chlorosomes were isolated from two types of green photosynthetic bacteria Chlorobium (Chl.) tepidunt and Chlorojflexus (Cfl.) aurantiacus and were individually imaged in the frozen medium. Each fluorescence spot observed mainly came from a single chlorosome and was ascribable to self-aggregates of bacteriochlorophyll (BChl) c molecules as core parts of chlorosomes. A three-dimensional distribution of fluorescence of single chlorosomes was analyzed, and the number of chlorosomes in a volume of 54 000 mu M3 was counted directly. On the basis of the results, averaged numbers of the BChl c molecules contained in a single chlorosome of Chl. tepiduin and Cfl. aurantiacus were determined to be 1.4 x 10(5) and 9.6 x 10(4), respectively. The present numbers are almost comparable to those estimated by other methods (MartinezPlanells et al., Photosynth. Res. 2002, 71, 83 and Montano et al., Biophys. J. 2003, 85, 2560).