The dicistronic assay for internal ribosome entry site (IRES) activity is the most widely used method for testing putative sequences that may drive cap-independent translation initiation. This assay typically involves the transfection of cells with dicistronic DNA test constructs. Many of the reports describing eukaryotic IRES elements have been criticized for the use of inadequate methods for the detection of aberrant RNAs that may form in transfected cells using this assay. Here we propose the combined use of a new RNAi-based method together with RT-PCR to effectively identify aberrant RNAs. We illustrate the use of these methods for analysis of RNAs generated in cells transfected with dicistronic test DNAs containing either the hepatitis C virus (HCV) IRES or the X-linked inhibitor of apoptosis (XIAP) cellular IRES. Both analyses indicated aberrantly spliced transcripts occurred in cells transfected with the XIAP dicistronic DNA construct. This contributed to the unusually high levels of apparent IRES activity exhibited by the XIAP 5' UTR in vivo. Cells transfected directly with dicistronic RNA exhibited much lower levels of XIAP IRES activity, resembling the lower levels observed after translation of dicistronic RNA in rabbit reticulocyte lysates. No aberrantly spliced transcripts could be detected following direct RNA transfection of cells. Interestingly, transfection of dicistronic DNA or RNA containing the HCV IRES did not form aberrantly spliced transcripts. These observations stress the importance of using alternative test procedures (e.g., direct RNA transfection) in conjunction with a combination of sensitive RNA analyses for discerning IRES-containing sequences in eukaryotic mRNAs.