Several recent publications have explored cap-independent translation through an internal ribosome entry site (IRES) in the 5'-UTR of the mRNA encoding the cyclin-dependent kinase inhibitor p27. The major experimental tool used in these reports was the use of bicistronic reporter constructs in which the 5'-UTR was inserted between the upstream and downstream cistrons. None of these reports has completely ruled out the possibility that the 5'-UTR has either cryptic promoter activity or a cryptic splice acceptor site. Either of these possibilities could result in expression of a monocistronic mRNA encoding the downstream cistron and false identification of an IRES. Indeed, Liu et al. recently published data suggesting that the p27 5'-UTR harbors cryptic promoter activity which accounts for its putative IRES activity. In this report, we have explored this potential problem further using promoterless bicistronic constructs coupled with RNase protection assays, siRNA knockdown of individual cistrons, RT-PCR to detect mRNA encoded by the bicistronic reporter with high sensitivity, direct transfection of bicistronic mRNAs, and insertion of an iron response element into the bicistronic reporter. The results do not support the conclusion that the p27 5'-UTR has significant functional promoter activity or cryptic splice sites, but rather that it is able to support cap-independent initiation of translation.