Mammalian ribosomal DNA (rDNA) transcription has a certain species specificity such that, both in vivo and in vitro, human rDNA cannot be transcribed by mouse machinery and vice versa. This is due to a species-dependent transcription factor, TFID (Y. Mishima, I. Financsek, R. Kominami, and M. Muramatsu, Nucleic Acids Res. 10:6659-6670, 1982). On the basis of the information obtained from 5' and 3' substitution mutants, we prepared a chimeric gene in which the mouse sequence from positions -32 to -14 was inserted into the corresponding location of the human rDNA promoter. The chimeric gene could be transcribed by mouse extracts nearly as efficiently as the wild-type mouse promoter. The chimeric gene could also sequester transcription factor TFID at an efficiency similar to that for the mouse promoter. Partially purified mouse TFID that could not protect the human rDNA promoter against DNase I produced a clear footprint on this chimeric gene that was similar to that on mouse rDNA promoter. The basic structure of the mouse rDNA core promoter is discussed in relation to the interaction with TFID.