We constructed a series of BspMI cassettes that simplify the introduction of specific point mutations in the polymerase domain of human immunodeficiency virus type 1 reverse transcriptase. A series of point mutants were constructed by using these cassette vectors. The RNA-dependent DNA polymerase and RNase H activities of 20 point mutations in the conserved portion of the polymerase domain were assayed. All the mutations analyzed are conservative substitutions of evolutionarily conserved amino acids. The mutations were divided into four classes. The first class has little effect on either polymerase or RNase H activity. The second class affects RNase H but not polymerase activity, while the third class has a normal RNase H activity with diminished polymerase activity. The fourth class affects both activities.