The pituitary plays significant roles in the regulation of physiological processes. In the current study, expressed sequence tag data was obtained for 1,920 clones from a normalized mixed-sex pituitary cDNA library. From these 3,840 sequences, a total of 524 contigs were assembled and 1,256 unique singletons identified. Assignment of functional annotation was performed through BLAST and gene ontology term assignment. Through in silico comparative mapping homologs were identified for 354 of the unigene sequences. These data provide the first functional information on many of the transcripts present in the rainbow trout pituitary.