Transcriptional elongation by RNA polymerase II has been well studied in vitro, but understanding of this process in vivo has been limited by the lack of a direct and specific assay. Here, we designed a specific assay for transcriptional elongation in vivo that involves an artificial arrest (ARTAR) site designed from a thermodynamic theory of DNA-dependent transcriptional arrest in vitro. Transcriptional analysis and chromatin immunoprecipitation experiments indicate that the ARTAR site can arrest Poi II in vivo at a position far from the promoter. TFIIS can counteract this arrest, thereby demonstrating that it possesses transcriptional antiarrest activity in vivo. Unexpectedly, the ARTAR site does not function under conditions of high transcriptional activation unless cells are exposed to conditions (6-azauracil or reduced temperature) that are presumed to affect elongation in vivo. Conversely, TFIIS affects gene expression under conditions of high, but not low, transcriptional activation. Our results provide physical evidence for the discontinuity of transcription elongation in vivo, and they suggest that the functional importance of transcriptional arrest sites and TFIIS is strongly influenced by the level of transcriptional activation.