We have previously shown that of the two functional domains which constitute the Xenopus 5S gene promoter the common, conserved box A element is directly involved in the binding of the common transcription factor IIIC. Here, we describe the investigation of the role of the 5S gene specific promoter element, box C, in transcription factor binding. Analysis of 22 different single site basepair changes reveals that mutations created within the 5'-region of this segment interfere with transcription due to a reduced affinity for TFIIIA, whereas sequence alterations introduced into the 3'-region of the same element similarly inhibit transcription, but do not result in a measurable defect in TFIIIA binding. Instead, they clearly reduce the affinity for TFIIIC. DNase I protection experiments with TFIIIA on 5S mutants which have an increased spacing of box A and box C demonstrate that TFIIIA recognizes a specific box A sequence element and that the factor has to be properly aligned on the DNA template in order to allow stable complex formation with TFIIIC to proceed. The structural and functional organization of protein binding signals on the 5S gene internal control region will be discussed in regard to these results.