The RNA-dependent protein kinase (PKR) is regulated by the binding of double-stranded RNA (dsRNA) or single-stranded RNAs with extensive duplex secondary structure. PKR has an RNA binding domain (RBD) composed of two copies of the dsRNA binding motif (dsRBM). The dsRBM is an alpha-beta-beta-beta-alpha structure present in a number of proteins that bind RNA, and the selectivity demonstrated by these proteins is currently not well understood. We have used affinity cleavage to study the binding of PKR's RBD to RNA. In this study, we site-specifically modified the first dsRBM of PKR's RBD at two different amino acid positions with the hydroxyl radical generator EDTA.Fe. Cleavage by these proteins of a synthetic stem-loop ligand of PKR indicates that PKR's dsRBMI binds the RNA in a preferred orientation, placing the loop between strands beta1 and beta2 near the single-stranded RNA loop. Additional cleavage experiments demonstrated that defects in the RNA stem, such as an A bulge and two GA mismatches, do not dictate dsRBMI's binding orientation preference. Cleavage of VA(I) RNA, an adenoviral RNA inhibitor of PKR, indicates that dsRBMI is bound near the loop of the apical stem of this RNA in the same orientation as observed with the synthetic stem-loop RNA ligands. This work, along with an NMR study of the binding of a dsRBM derived from the Drosophila protein Staufen, indicates that dsRBMs can bind stem-loop RNAs in distinct ways. In addition, the successful application of the affinity cleavage technique to localizing dsRBMI of PKR on stem-loop RNAs and defining its orientation suggests this approach could be applied to dsRBMs found in other proteins.