Several types of small RNAs have been proposed as gene expression repressors with great potential for use in gene therapy. RNA polymerase III (pol III) provides an ideal means of expressing small RNAs in cells because its normal products are small, highly structured RNAs that are found in a variety of subcellular compartments. We have designed cassettes that use human pol III promoters for the high-level expression of small RNAs in the cytoplasm, nucleoplasm, and nucleolus. The levels and subcellular destinations of the transcripts are compared for transcripts expressed using the U6 small nuclear RNA (snRNA), 5S ribosomal RNA (rRNA), and the 7SL RNA component of the signal recognition particle. The most effective location for a particular inhibitory RNA is not necessarily predictable; thus these cassettes allow testing of the same RNA insert in multiple subcellular locations. Several small interfering RNA (siRNA) inserts were tested for efficacy. An siRNA insert that reduces lamin expression when transcribed from the U6 snRNA promoter in the nucleus has no effect on lamin expression when transcribed from 5S rRNA and 7SL RNA-based cassettes and found in the nucleolus and cytoplasm. To test further the generality of U6-driven siRNA inhibitors, siRNAs targeting HIV were tested by co-transfection with provirus in cell culture. Although the degree of HIV-1 inhibition varied among inserts, results show that the U6 cassette provides a means of expressing an siRNA-like inhibitor of HIV gene expression.