A total of 77 diaryl amidine derivatives were evaluated for their inhibitory effects on the DNA polymerase (reverse transcriptase) activity of Moloney murine leukemia virus. The amidino-substituted aryl moieties of the compounds consisted of phenyl, indole, indene, benzofuran, benzo[ b] thiophene, or benzimidazole. Several of these compounds were found to inhibit oncornavirus-directed DNA synthesis with an ID₅₀ of approximately 1 μg/mL. This ID₅₀ was comparable to that of ethidium bromide, a compound which is considered to be a very effective inhibitor of oncornaviral DNA polymerases. Structure-function analysis revealed that the inhibitory activity of the diaryl amidine compounds depended on the planarity of the molecule, the presence of both amidino (or, preferably, imidazolino) groups, the absence of a central bridge or, if present, a short and rigid chain connecting the two aryl moieties, and, finally, the nature of these aryl residues (preferably benzofuran or indole). Hence, compounds 18 (4',6-diimidazolino-2 phenylbenzo[ b] thiophene) and 48 [5-amidino-2-(5-amidino-2-benzofuranyl)indole] featured among the most potent inhibitors of oncornavirus-directed DNA synthesis. For some representative diaryl amidines the kinetics of inhibition of viral DNA synthesis was investigated with (A)ₙ·oligo(dT) as template-primer and dTTP as substrate and assuming a quasi-steady-state reaction with a single substrate (dTTP), a single enzyme (reverse transcriptase), and a single product [(A)ₙ·(dT)ₘ]. Inhibition was not exactly competitive with respect to (A)ₙ·oligo(dT), although a purely competitive type of inhibition may have been expected if the compounds simply blocked the binding site(s) of the template-primer for the enzyme.