The function of unknown genes is often inferred from comparisons to well-characterized homologs. In this paper, we show that, even if all of the homologs of a gene are unannotated, its function may be deduced through phylogenetic profiling. We have designed a series of algorithms that make functional predictions of genes based on orthology and set theory, but our approach to predicting gene function requires no previous knowledge of homolog function. With this technique, we successfully identified 94% of the clusters of orthologous groups that are known to be involved in flagella development or function. As a test, we removed the function of three putative flagellar genes that had been previously uncharacterized in Bacillus subtilis. We observed a motility phenotype for two of these three genes. Thus, these algorithms allow for high-throughput functional prediction of genes beyond that provided by simple orthology-based annotation endeavors.