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Deletions extending from the cro gene into boxA and nutR of the Rho-dependent tRi terminator of bacteriophage X have been generated and cloned between promoters and the galK gene of Escherichia coli on a multicopy plasmid. Terminators placed between the promoters and galK restrict transcription and expression of galK on these plasmids. However, when X N protein is provided, and if a functional N interaction site, nutR, is intact, transcription antitermination occurs and galK expression increases. Deletions into the nutR region affect the ability to antiterminate. From the results obtained we conclude that: (i) boxA, a site believed to bind host factors (Nus), is not required for transcription antitermination in this system; (ii) the host NusA function is required even in the absence of boxA; (idi) nutR is required for N antitermination; (iv) translation across the nutR sequence prevents Ndependent antitermination.The N gene product of bacteriophage X positively regulates phage early gene expression by antitermination of transcription at various terminator signals (see ref. 1). Salstrom and Szybalski (2) isolated a cis-acting mutation, nutL, in the PL operon that impairs N protein-mediated antitermination activity. A similar site of protein N action in the right operon of phage X, referred to as nutR, has also been identified on the basis of sequence homology to nutL (3), genetic deletion studies (4), and cloning experiments (5). In addition, an octamer sequence, CGCTCTTA, called boxA, located just promoter-proximal to nutR, has been implicated in Escherichia coli host protein NusA interaction (6,7). The host proteins involved in antitermination are a complex of factors including at least the nusA, nusB, and nusE gene products [see review by Friedman et al. (8) and ref. 9]. To reveal the individual cis-acting components that participate in interaction with various phage-and host-encoded factors, deletions were generated in vitro in boxA and nutR and assayed for their effect on antitermination activity in vivo.