We obtained genetic information on sexual reproduction of the Closterium peracerosum-strigosum-littorale complex, a unicellular charophycean alga. Normalized cDNA libraries were constructed from cells in the sexual reproduction process, and a total of 1190 5'-end expressed sequence tags were established. Since 604 of these ESTs were classified into 174 non-redundant sequences, these 1190 ESTs include 760 unique sequences. Similarity search against a public non-redundant protein database indicated that 390 unique sequences had significant similarity to registered sequences. Among these 390 sequences, 3 were identical to and 4 were homologous to previously identified sex-pheromone genes. According to our study, 370 of 760 unique sequences are likely to be novel transcripts. These cDNA clones and EST sequence information may would be helpful for future functional analyses using DNA array technologies.