The rat muscle creatine kinase (CKM) gene promoter is unusual since it is one of the few cellular promoters containing a p53 response element which is located proximally (bp -168 to -57) to the transcription start site. We have previously shown that p53wt transactivates transcription in vivo of rat CKM, in CV-1 monkey kidney cells, through this 112 bp promoter-proximal fragment which contains at least five degenerate p53-binding elements. In this report, we employed the gel-shift assay and demonstrated that recombinant, immunoaffinity-purified mouse p53wt binds to this 112 bp CKM sequence and activates the in vitro transcription of the proximal CKM promoter by nuclear extracts from CV-1 cells. Also, a competitor plasmid containing this 112 bp CKM fragment interferred with the in vivo transactivation of CKM by p53. This CKM fragment, when cloned upstream of the rat brain creatine kinase (CKB) promoter, mediated the p53 transactivation of CKB. Analyses of p53wt and a series of missense mutants (altered in conserved region II of p53) showed that binding of p53 to the CKM promoter was required but was not sufficient for transactivation. The results are discussed in relation to the possible role of p53wt in the expression of CKM in cell types which may not express the myogenic transcription factors.