Both qualitative and quantitative patterns of tissue-specific gene expression can be determined using gene profiling. Expressed sequence tag (EST) analysis is an efficient approach not only for gene discovery and examining gene expression, but also for development of molecular resources useful for functional genomics. As part of an ongoing transcriptome analysis of channel catfish (Ictalurus punctatus), EST analysis was conducted for gene annotations and profiling using a complementary DNA library developed from messenger RNA of the spleen. A total of 1204 spleen cDNA clones were analyzed. Of the 1204 clones, 665 clones (55.2%) were identified as orthologs of known genes from other organisms by BLAST searches and 539 clones (44.8%) as unknown gene clones. In total 147 novel genes were identified, and annotations were made to 118 of them. In addition, 389 novel EST clusters were identified. Expression profile was analyzed in relation to metabolic functional groups. A total of 28 known genes were involved in immune functions, of which 10 were identified for the first time in channel catfish. Microsatellite-containing clones were also identified that may be potentially useful for genome mapping. This work contributed to the Catfish Gene Index, and toward a Unigene set useful for functional genomics research concerning spleen gene functions in relation to disease defenses.