PURPOSE: To characterize gene expression patterns in various tissues of the zebrafish (Danio rerio) eye and identify zebrafish orthologs of human genes by expressed sequence tag (EST) analysis for NEIBank. METHODS: mRNA was extracted from adult zebrafish eye tissues, including lenses, anterior segments (minus lens), retinas, posterior segments lacking retinas, and whole eyes. Five different cDNA libraries were constructed in the pCMVSport6 vector. Approximately 4,000 clones from each library were sequenced and analyzed using various bioinformatics programs. RESULTS: The analysis yielded approximately 2,500 different gene clusters for each library. Combining data from the five libraries produced 10,392 unique gene clusters. GenBank accession numbers were identified for 37.6% (3,906) of the total gene clusters in the combined libraries and approximately 50% were linked to Unigene clusters in the current database. Several new crystallin genes, including two gammaN-crystallins, and a second major intrinsic protein (MIP) were identified in the lens library. In addition, a zebrafish homolog of cochlin (COCH), a gene that may play a role in the pathogenesis of human glaucoma, was identified in the anterior segment library. Surprisingly, no clear ortholog of the major retinal transcription factor Nrl was identified. CONCLUSIONS: The zebrafish eye tissue cDNA libraries are a useful resource for comparative gene expression analysis. These libraries will complement the cDNA libraries made for the Zebrafish Gene Collection (ZGC) and provide an additional source for gene identification and characterization in the vertebrate eye.