A simple method using molecular hybridization was devised to quantitatively measure chloroplast DNA synthesis in vivo. Total cellular DNA isolated from Nicotiana tabacum suspension cells, labeled with (3)H-thymidine, was hybridized to nitrocellulose membrane-bound cloned chloroplast DNA (ct DNA) fragments. Colorless, dark grown N. tabacum cells were found to contain approximately 3300-4800 chloroplast genome copies per cell, whereas light grown, green cells contain about 9500-12000 chloroplast genomes per cell. This difference in ct DNA levels suggests that the chloroplast genome is somewhat amplified during growth of the cells in the light. The hybridization technique was also used to measure the efficiency of hybridization between cloned spinach ct DNA and tobacco ct DNA. The two DNAs were found to cross-hybridize with an efficiency of 69-75%.