The genome of the human delta hepatitis agent is a circular, highly structured single-stranded RNA lacking regular runs of RNA-RNA duplex longer than 15 bp. We have tested the ability of delta agent RNA to participate in reactions with a protein containing a motif which confers the ability to bind double-stranded RNA (dsRNA). Surprisingly, highly purified delta agent RNA preparations from which all traces of contaminating dsRNA have been removed activate PKR, the dsRNA-dependent protein kinase activity of mammalian cells (also known as DAI, P1-eIF-2, and p68 kinase). This behavior is in marked contrast to the interaction of PKR with a number of other highly structured viral single-stranded RNAs, which inhibit, rather than stimulate, activation of this kinase. PKR activation leads to inhibition of protein synthesis in the rabbit reticulocyte lysate system. Paradoxically, delta RNA failed to elicit the expected PKR-mediated inhibition of cell-free translation. Instead, delta RNA interfered with PKR activation and the translational block induced by dsRNA. We conclude that the interaction of PKR and delta agent RNA may represent a new category of protein-RNA interactions involving the dsRNA binding motif.