A factor ( TFIIIA ) specifically required for transcription of the 5 S RNA gene has been physically and enzymatically characterized in its native state. TFIIIA is a monomer of 38,600 Da with a Stokes radius of 34 A and a sedimentation coefficient of 2.8 S. A calculated frictional ratio of 1.53 indicates that it is highly asymmetric, and suggests the possibility that as little as 1 molecule bound/5 S RNA gene is sufficient to account for the previously documented interaction with the promoter region of the gene. In addition, TFIIIA does not contain any detectable ATPase or protein kinase activity, and its ability to be used in transcription is inhibited by N-ethylmaleimide. Incubation of a 5 S gene-containing plasmid with a molar excess of TFIIIA results in the formation of a complex which can be separated from unbound TFIIIA by gel exclusion chromatography. The isolated complex displays a TFIIIA :5 S gene stoichiometry of 1:1, is fully functional when complemented with other transcription components, and is directly dependent upon the presence of a 5 S gene for its formation. Further analyses of requirements for complex formation indicate that the presence of 5 S RNA in the prechromatographic incubation is required to eliminate nonspecific interactions of TFIIIA while at the same time allowing retention of its gene-specific interaction.