Alu and 7SL RNA gene sequences were tested for the potential to regulate mammalian cell growth by introducing these sequences into HeLa cells in a coupled DEAE-dextran transfection/affinity cell sorting system. Both Alu and 7SL RNA genes mediated inhibition of [3H]thymidine and [35S]methionine incorporation in recipient cells. In addition, short term growth curves were performed on calcium phosphate/DNA cotransfected, affinity-purified cells. This second assay revealed that transfected Alu and 7SL RNA can also cause suppression of HeLa cell proliferation. To investigate whether transcription or polymerase III (pol III) transcription factor binding was required for inhibitory activity, mutations were introduced into RNA pol III B block promoter elements in each of these genes. Suppression of [3H]thymidine incorporation was dependent on the presence of this pol III element in both genes; likewise, 7SL RNA-mediated growth suppression required the presence of the pol III B block promoter element. The results of this study indicate that Alu and 7SL RNA gene sequences interact with cellular factors that are important for HeLa cell proliferation and suggest that these pol III-transcribed elements may be involved in the regulation of cellular growth.