A set of partially overlapping DNA restriction fragments that support promoter-dependent transcription of the tRNATyr1 gene of Escherichia coli has been used to study site-specific termination in vitro. Transcription termination occurs at a specific site 224-226 nucleotides beyond the end of the structural gene and is completely dependent on rho-factor. Certain features of this site suggest differences from other termination sites previously studied. A role for specific sequence recognition is suggested.