We have isolated and mutagenized a DNA fragment from Saccharomyces cerevisiae that specifies mRNA 3' end formation for the convergently transcribed CYC1 and UTR1 genes. An in vivo plasmid supercoiling assay previously showed that this fragment is a transcriptional terminator, and "run-on" assays shown here are consistent with this interpretation. The poly(A) sites in the mRNAs formed by the fragment are the same whether the fragment resides at the native location or at a heterologous location. No single linker substitution abolishes the fragment's activity, whereas certain large, nonoverlapping deletions have strong, deleterious effects. Therefore, the yeast terminator behaves more like rho-dependent bacterial terminators than terminators of higher eukaryotes. That a number of deletions or substitutions have different effects in the two orientations suggests that the fragment contains the sequences of two, unidirectional terminator elements.