Two cDNA libraries from Takifugu rubripes spermatozoa and eggs were constructed and a total of 620 expressed sequence tag (EST) clones were generated from the two libraries: 300 clones are from the spermatozoa library and 320 clones are from the eggs library. The most abundant cDNA clones in the two libraries were identified. A total of 207 'contigs' (or single) EST clones were found to share significant sequence identity with known sequences in the GenBank database, representing at least 51 different genes. In order to understand the two types of germ cells further, the expression profiles of the identified clones in these cDNA libraries were analyzed. Furthermore, the presence of specific messenger RNAs in the spermatozoa and eggs has been demonstrated with BLAST analysis; the spermatozoa and egg library can supply unique and novel cDNA sequences in the Takifugu rubripes EST project. Another aim of this study is to identify cDNA clones that can be used as molecular markers for the analysis of the spermatogenesis and oogenesis in Takifugu rubripes. Six potential clones (S1-3 from spermatozoa and E1-3 from eggs) were selected to analyze their expression patterns by reverse transcription (RT)-PCR analyses. Half of these showed a specific expression in the expected tissue. Two of the clones were found by RT-PCR and in situ hybridization to be expressed specifically in the testis or ovary, and they maybe suitable molecular markers for the analysis of spermatogenesis and oogenesis.