A fluorescence polarization (FP) microplate assay suitable for screening compounds against the ribonuclease H (RNase H) activity of HIV-1 reverse transcriptase has been developed. This homogeneous assay uses a hybrid 18-mer DNA/RNA duplex substrate composed of an RNA oligonucleotide labeled with 6-carboxytetramethyl rhodamine at the 3' end that is annealed to a complementary unlabeled DNA strand. The labeled RNA/DNA duplex demonstrated Michaelis-Menten kinetics with a Km value of 9.6+/-2.8 nM. Substrate cleavage by RNase H to produce small RNA fragments (1-4 mer) resulted in a large change in the measured FP value. This FP assay was amenable to kinetics protocols as well as stopped endpoint measurements. When using the latter for conducting robotics runs, Z' values greater than 0.8 typically were observed. The stopped endpoint FP assay was used successfully in a high-throughput screening campaign to screen 1.8 million compounds for RNase H inhibition.