The temporal evolution of pigment composition during greening of etiolated barley leaves was investigated by reversed-phase HPLC with particular attention to chlorophyll (Chl) a' (C13(2) epimer of Chl a), which had been detected by ourselves in photosystem (PS) I particles. At early stages of greening, the Chl a'/Chl a molar ratio rapidly increased to a level more than twice that in mature leaves, then gradually leveled off, accompanied by a growth of the Chl b/Chl a ratio, to the mature level. After 3 h of illumination, the temporal evolution of the Chl a'/Chl a molar ratio nearly paralleled that of the P700/Chl a ratio with a stoichiometry Chl a'/P700 approximately equal to 2: this strongly suggests that Chl a' is biosynthesized as a constituent of the PS I reaction center complex.