Cultured leaf disks from young spinach leaves have been used to study chloroplast DNA synthesis in relation to chloroplast growth and replication. Autoradiographs prepared from thin sections show that labelling throughout disks is uniform. We consider that cultured leaf disks serve as a model system for studies of cell expansion and chloroplast replication during the development of spinach leaf cells. Leaf disks from young spinach leaves were incubated continuously for 5 days in a medium containing [3H]thymidine. They were harvested daily and light microscope autoradiographs of separated cells prepared. In the expanding cells of disks all chloroplasts divide and the synthesis of chloroplast DNA as measured from grain counts was correlated with the rate of chloroplast formation. A doubling of chloroplast numbers was associated with a doubling of chloroplast silver grains, which is consistent with a duplication of chloroplast DNA during the chloroplast division cycle. When disks were grown in green light, chloroplast replication but not chloroplast growth ceased in 3-4 days. The large chloroplasts of disks grown in green light appeared locked in a dumb-bell shaped configuration. Light microscope autoradiographs have established that these chloroplasts can synthesize DNA. These chloroplasts divided when the disks were exposed to high intensity white light and again it appears that all chloroplasts in the one cell divide. The chloroplasts of disks grown in green light for periods up to 9 days became uneven in size although all were dumb-bell shaped. We suggest that the size differences represent ploidy classes. Chloroplast replication in dicotyledons has been discussed in relation to the division and growth of leaf cells.