Adenovirus VA RNA, maintains protein synthesis by preventing activation of the double-stranded RNA (dsRNA)-dependent protein kinase DAI. There appears to be a single binding site for dsRNA on DAI, and this site is blocked by VA RNA,. VA RNA, binds to purified DAI and can be cross-linked to the enzyme by UV irradiation. To determine the relationship between DAI binding and VA RNA, structure and function, we examined the binding abilities of wild-type and mutant VA RNAs. In several cases, the ability to bind DAI efficiently in vitro did not correlate with function in vivo. Secondary structure analysis suggested that efficient binding requires an apical stem-loop structure, whereas inhibition of DAI activation requires the central domain of the VA RNA molecule. We propose that the duplex stem permits VA RNA to interact with the dsRNA binding site on DAI and inhibits activation by juxtaposing the central domain of the RNA with the enzyme’s active site.