We describe the sequence and characterization of the Bacillus subtilis flhF gene. flhF encodes a basic polypeptide of 41 kDa that contains a putative GTP-binding motif. The sequence of FlhF reveals a structural relationship to two Escherichia coli proteins, Ffh and FtsY, as well as to other members of the SRP54 family, in a domain presumed to bind GTP. flhF is located in a large operon consisting of chemotaxis and flagellar genes. Cells deficient in flhF are nonmotile. Through the use of anti-flagellar antibodies we have established that flhF is a flagellar (fla) gene. Thus, flhF is a unique flagellar gene in that it encodes a GTP-binding protein with similarities to members of the SRP54 family of proteins. These data suggest that flagellar biosynthesis in B. subtilis requires GTP.