The in vitro recombination of deletion mutants of the Xenopus borealis somatic 5 S RNA gene allows construction of mutants containing substitutions of synthetic linker DNA for blocks of 9 or 10 nucleotides within the intragenic control region that binds transcription factor A. Two of the mutants containing exact linker replacements without insertion or deletion of nucleotides together alter 12 of 21 base pairs within the center of the control region. Both of these mutants bind factor A in an essentially normal pattern and support transcription, suggesting that a remarkable sequence variability can be tolerated in the center of the control region. The data obtained with these linker substitution mutants, along with data obtained previously with unidirectional deletion mutants, indicate that two short sequences at the boundaries of the control region serve as important recognition sites for the transcription factor. Additional closely related mutants containing slight deletions or insertions of form 2 to 10 base pairs do not bind factor A normally and do not support transcription in vitro. Thus, precise relative spacing of the boundaries of the control region is of critical importance for transcription.