In an attempt to isolate cDNA clones for dark-inducible chloroplast proteins, we screened a cDNA library which was prepared from radish cotyledons by a two-step method. The source plants were grown under continuous light for 14 d and kept in darkness for 24 h. One of the selected clones, S2D12, corresponded to the din1 gene which we previously reported as a dark-inducible, senescence-associated gene [Azumi and Watanabe (1991) Plant Physiol. 95:577]. A 22 kDa polypeptide was produced from the cDNA in an in vitro expression system in the presence of [35S]methionine. This polypeptide was capable of being imported by isolated chloroplasts, processed to a smaller mature form and localized in the stromal fraction. As the amino acid sequence of the putative mature protein has no homology to any known chloroplast protein, din1 was suggested to be the first gene for a chloroplast protein which is negatively controlled by light. The putative mature protein has similarity to sulfide dehydrogenase from Wolinella succinogenes and other small stress proteins; glpE and pspE from Escherichia coli and hsp67B2 from Drosophila melanogaster.