Insulin-like growth factor-II (IGF-II) mRNAs are subject to site-specific endonucleolytic cleavage in the 3′ untranslated region (UTR), rendering an unstable 5′ cleavage product containing the coding region and a very stable 3′ cleavage product of 1.8 kb consisting of the 3′-UTR sequence and the poly(A) tail. Previously, it was established that two widely separated elements in the 3′-UTR (elements I and II), that can form a duplex structure, are necessary and sufficient for cleavage. To further investigate the sequence and secondary structure requirements for cleavage, we have introduced a number of mutations around the cleavage site and assayed their effects on cleavage. Several recognition determinants involved in the endonucleolytic cleavage of IGF-II mRNAs were identified. Mutational analysis around the cleavage site revealed that cleavage is sequence specific and that the cleavage site must be in a single-stranded conformation to allow efficient cleavage. In addition, we have identified an accessory protein that specifically interacts with a stem-loop structure located 133 to 73 nt upstream of the cleavage site.