Three different DNA polymerase species can be identified by phosphocellulose chromatography following treatment of the avian myeloblastosis virus αβ DNA polymerase with 1,4-dioxane: (1) α DNA polymerase, (2) residual αβ DNA polymerase, and (3) a species enriched for β DNA polymerase. Binding studies with the major species of primer RNA (tRNA TrP) involved in the initiation of RNA-directed DNA synthesis in vitro, were carried out with these three DNA polymerase species. Both αβ and the β DNA polymerase enriched species bound tightly to [ 32P]tRNA Trp as demonstrated by the ability of both enzymes to exclude tRNA Trp on Sephadex G-75 columns and by increasing the sedimentation rate of tRNA Trp in glycerol gradients. Neither enzyme required divalent metal ion for binding to tRNA Trp at 0°. The binding capacity of these two enzymes to tRNA Trp was significantly reduced in the presence of MgCl 2. This reduction in binding capacity was most likely due to conformational changes of tRNA Trp resulting from selective binding with Mg 2+. α DNA polymerase, even when present in a 1000-fold molar excess with respect to tRNA Trp, did not bind to this primer RNA irrespective of whether or not divalent metal ion was present. These results suggest that the β subunit in αβ DNA polymerase is required for effective binding of the holoenzyme to tRNA Trp.