The secretome is a functionally rich proteome subset, including cellular membrane and extracellular proteins processed through the secretory pathway. In this study, Danio rerio and Homo sapiens RefSeq proteins were analyzed with SignalP, TargetP, Phobius, and pTarget algorithms. About 16.5% of the zebrafish proteome and 17.0% of the human proteome possessed predicted N-terminal signal sequences. Nearly half of these proteins were subsequently classified as soluble, as they lacked predicted transmembrane domains. The soluble proteins were further subclassified, predicting 1345 (3.8%) zebrafish and 1207 (3.2%) human proteins as extracellular. Comparison of the zebrafish and human soluble secretome proteins identified 372 as orthologs, on the basis of reciprocal BLAST best hits. The computational characterization of the zebrafish proteins found many more members of the secretome than annotated in the SwissProt database. Only 180 of the 2078 zebrafish SwissProt protein entries, and 995 of the 19,294 human SwissProt protein entries were annotated with secreted protein locales. A specific investigation of the fibroblast growth factor and matrix metalloproteinase (MMP) protein families confirmed the prediction data and generated annotation of three additional putative MMP zebrafish proteins. This study presents the first known published description of the zebrafish secretome since the completion of the zebrafish genome sequencing project.