To collect information on gene expression during the dark period in the luminous dinoflagellate Lingulodinium polyedrum, normalized complementary DNA (cDNA) libraries were constructed from cells collected during the first hour of night phase in a 12:12 h light–dark cycle. A total of 4324 5′-end sequence tags were isolated. The sequences were grouped into 2111 independent expressed sequence tags (EST) from which 433 groups were established by similarity searches of the public nonredundant protein database. Homology analysis of the total sequences indicated that the luminous dinoflagellate is more similar to land plants and animals (vertebrates and invertebrates) than to prokaryotes or algae. We also isolated three bioluminescence-related (luciferase and two luciferin-binding proteins [LBP]) and 37 photosynthesis-related genes. Interestingly, two kinds of LBP genes occur in multiple copies in the genome, in contrast to the single luciferase gene. These cDNA clones and EST sequence data should provide a powerful resource for future genome-wide functional analyses for uncharacterized genes.