We have characterized the functional attributes of a 211-base pair region containing the Escherichia coli tryptophan operon rho-dependent terminator trp t', utilizing a series of constructs that alter the orientation, location, or extent of the trp t' sequences with respect to the trp promoter. In each instance, the extent of the rho-dependent response was monitored in vivo by read-through expression into a distal galactokinase gene and was compared with the results of transcription assays in vitro. As expected, transcription termination in vivo is dependent on the proper orientation of the terminator, and a tandem repeat of the terminator increases termination proportionally. Placing the terminator fragment only 14 nucleotides from the promoter does not affect termination significantly, supporting the belief that sequences outside of the 211-base pair fragment itself are dispensable. One construct, which lacks 116 base pairs, including the region encoding the normal RNA end points, still reduces galK activity in vivo and terminates transcription in vitro. Our results indicate that the rho response depends primarily on sequences in this 95-base pair segment, causing RNA polymerase to terminate transcription in a region 15-45 nucleotides further downstream.