Confocal laser scanning microscopy was used to study the distribution of the smallest detectable autofluorescing, chlorophyll-bearing structures in fresh, 40 microm thick longitudinal sections of the shoot apex of four dicotyledonous plants (Arabidopsis thaliana, Nicotiana glauca, Lupinus alba, and Spinacia oleracea). In all species, the smallest chlorophyll-bearing particles were found in the outermost cell layers (L1 and L2) of the shoot apex. Their distribution between these layers differed in each species. The smallest such particles were about 0.5-1.0 microm in maximum dimension, approximating the size of a single granum in the developing leaf. Their size and abundance increased with increasing cell age and distance from the peak of the apex. Immediately beneath the L1 and L2 layers was a zone largely devoid of these particles. Below this nonfluorescing zone, in the region where the derivatives of the meristematic zone differentiate into cells of the central pith region, the size and abundance of the chlorophyll-bearing particles increased progressively with increasing distance from the nonfluorescing zone. The presence of these small autofluorescing particles in the L1 and L2 cell layers of the shoot apex places the development of photosystem II fluorescence at an earlier stage of leaf development than previously observed. The use of confocal laser scanning microscopy to study unfixed sections provides another useful metabolic marker for mapping patterns of differentiation and development in the cells of the shoot apex.