We analyzed the chlorophyll fluorescence parameters in a 3D cellular arrangement in vivo by using a modified Nipkow disk-type confocal laser scanning microscope (CLSM). We first defined the 3D values of Phi(PSII) (photochemical yield of PSII) and NPQ (non-photochemical quenching) in mesophyll, epidermal and guard cell chloroplasts from the leaf surface to several tens of microns in depth. We also used this CLSM method to analyze the relationships between actinic light intensity and the chlorophyll fluorescence parameters for Boston fern and broad bean leaf specimens. As the actinic light intensity increased, the mean Phi(PSII) values decreased and the NPQ values increased in all chloroplasts of Boston fern and broad bean leaf. These values differed with cell type and species. The Boston fern chloroplasts had lower Phi(PSII) values than the broad bean chloroplasts, and vice versa for the NPQ values. The Phi(PSII) values of Boston fern chloroplasts decreased in the order mesophyll, epidermal and guard cell chloroplasts. The NPQ values decreased in the order guard cell, mesophyll and epidermal chloroplasts, except at 12 micromol m(-2) s(-1) actinic light, when the mesophyll value was slightly lower than that of the epidermis. The trend in the Phi(PSII) and NPQ values of broad bean mesophyll and guard cell chloroplasts was opposite to that of Boston fern chloroplasts. As 3D CLSM can provide the Phi(PSII) and NPQ values of each chloroplast in a 3D cellular arrangement, this method has potential for investigating differences in the functions of chloroplasts in vivo.