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A subtractive cDNA library was made corresponding to mRNAs expressed in the neural complex relative to those expressed in the pharynx of adults of the ascidian Ciona intestinalis. Determination and comparison of expressed sequence tags (ESTs) of a set of 1,527 randomly selected clones demonstrated that they represent 832 independent sequences. Five hundred seventy-two of the clones contained amino-acid-encoding sequences. BLASTX analyses showed that 342 of the 572 clones were strong matches (P<10(-7)) to previously identified proteins, while the remaining 230 fell into the "no match" category. Among the clones matching previously identified proteins, about 80 clones represented proteins that are involved in the formation, maintenance of the structure, and function of the nervous system: 22 proteins are associated with signal transduction, five proteins are related to the synapse, 11 to transcription factors, nine to transporters, five to enzymes, and 13 to extracellular matrix and cytoskeletal components, and six to apoptosis. In addition, sequence information for genes associated with the immune system and for genes encoding proteins with interesting functions were obtained. These data provide cues for further studies on genes that are expressed in and function in the ascidian nervous system.