We report on the in silico-initiated cloning and molecular characterization of CTXN3 (cortexin 3), a new human gene that was specifically expressed in the kidney and brain due to tissue-specific alternative exon 1 usage, on chromosome 5q23.2 using digital gene expression displayer (DGED) and a novel in silico cloning approach based on both expressed sequence tags (ESTs) and genomic sequence. The gene CTXN3 included 3 exons and spanned an approximate 9.6-kb region of human chromosome 5q23. Two alternative transcript variants (GenBank accession nos. AB219764 and AB219832) were 1660 and 1458 bp long, respectively, encoding for an 81-amino acid protein with a predicted molecular weight of 8933.4 Da. The predicted human CTXN3 protein had 43% identity with function-unknown protein cortexin, which showed brain-specific expression. Further analysis of the encoded protein using PSORT II, TMpred, and PSIPRED programs demonstrated a putative single membrane-spanning domain in the middle of the CTXN3 amino acid sequence, indicating that it might be an integral membrane protein which may mediate extracellular or intracellular signaling of the kidney or brain. Analysis of the predicted CTXN3 orthologs from different species showed that these proteins are highly conserved in vertebrates. In conclusion, a combination of bioinformatics and molecular approaches is useful in the identification of genes expressed in specific tissues. Selective expression of CTXN3 in the kidney and brain, the amino acid identity to cortexin, and its high conservation among different species indicate that CTXN3 may be involved in a process specifically restricted to kidney and brain tissue function.