RNA secondary structure analysis was performed to understand the translation function of the adenovirus tripartite leader, a 200-nucleotide 5' noncoding region found on all late viral mRNAs. The tripartite leader facilitates the translation of viral mRNAs at late but not early times after infection and eliminates the normal requirement for the eukaryotic initiation factor 4F or cap binding protein complex. Secondary structures were determined by probing 5' or 3' end-labeled tripartite leader RNAs under nondenaturing conditions with various single strand-specific nucleases, and the information was used to generate a potential model structure. The resulting structure is attractive since it may explain the unusual translation behavior conferred by the tripartite leader. We demonstrate that the first leader segment is predominantly single-stranded, a property consistent with the ability to enhance translation and provide independence from cap binding protein complex. In contrast, the remaining two leader segments form a moderately stable base-paired structure, except for a large hairpin loop. To confirm these findings, the secondary structure of the tripartite leader was also probed when it was attached to a large segment of a messenger RNA and was found to be very similar to that of the individual leader RNA. These findings suggest several possible mechanisms to account for the translation activity of the tripartite leader.