Despite decades of investigation, the primary site of action of the prototypical inducers of differentiation, dimethylsulfoxide and hexamethylene bisacetamide (HMBA), has not been delineated. During studies designed to analyze cis-acting elements responsible for induction of stage-specific globin genes, we discovered the capacity of HMBA and dimethylsulfoxide to enhance the expression of transiently transfected reporter genes derived from globin and nonglobin gene promoters, prominently in nonerythroid 3T3 Swiss cells. The action of HMBA and dimethylsulfoxide in the transient transfection system resembled that of the inhibitor of histone deacetylases (HDACs), trichostatin A (TSA), in that the three agents enhanced reporter gene expression (a) regardless of the promoter employed, (b) with similar kinetics and (c) with an increase in the steady-state level of reporter mRNA. Transiently transfected DNA was assembled rapidly into a chromatinized structure in 3T3 cells, suggesting that transcription of reporter genes was at least in part repressed by chromatin organization. Nuclear run-on analyses indicated that dimethylsulfoxide and HMBA enhanced transcriptional initiation of the reporter and p21/WAF1/Cip1 genes. In contrast, TSA produced negligible effects on nuclear run-on transcription of these genes. HMBA and dimethylsulfoxide did not change the acetylation, phosphorylation, or methylation status of histones and did not activate stably transfected genes. Despite these differences, the three agents modulated the expression of common sets of cellular genes and induced differentiation or apoptosis in intact cells. The findings imply that HMBA and dimethylsulfoxide modulate transcription by a mechanism independent of histone acetylation.