The c-myc mRNA coding region determinant-binding protein (CRD-BP) has high affinity for the coding region determinant (CRD) of c-myc mRNA. Such affinity is believed to protect c-myc CRD from endonucleolytic attack. We have recently purified a mammalian endoribonuclease which can cleave within the c-myc CRD in vitro. The availability of this purified endonuclease has made it possible to directly test the interaction between CRD-BP and the endonuclease in regulating c-myc CRD RNA cleavage. In this study, we have identified the coding region of MDR-1 RNA as a new target for CRD-BP. CRD-BP has the same affinity for c-myc CRD nts 1705–1886 and MDR-1 RNA nts 746–962 with Kd of 500 nM. The concentration-dependent affinity of CRD-BP to these transcripts correlated with the concentration-dependent blocking of endonuclease-mediated cleavage by CRD-BP. In contrast, three other recombinant proteins tested which had no affinity for c-myc CRD did not block endonuclease-mediated cleavage. Finally, we have identified RNA sequences required for CRD-BP binding. These results provide the first direct evidence that CRD-BP can indeed protect c-myc CRD cleavage initiated by an endoribonuclease, and the framework for further investigation into the interactions between CRD-BP, c-myc mRNA, MDR-1 mRNA and the endoribonuclease in cells.