Vaccinia virus nucleoside triphosphate phosphohydrolase I (NPH I) is an essential early gene transcription termination factor. The C-terminal end of NPH I binds to the N-terminal end of the H4L subunit (RAP94) of the virion RNA polymerase. This interaction is required for transcription termination and transcript release. To refine our understanding of the specific amino acids in the C-terminal end of NPH I involved in binding to H4L, and to develop a collection of mutations exhibiting various degrees of activity to be employed in in vivo studies, we prepared a set of short deletions, and clustered substitutions of charged amino acids to alanine, or bulky hydrophobic amino acids to alanine mutations. These NPH I mutant proteins were expressed, purified, and tested for ATPase activity, binding to H4L, and transcription termination activity. Most mutations in amino acids 609 to 631 exhibited reduced activity. Deletion of the terminal five amino acids (627-631), or substitution of Y(629) with alanine or glutamic acid, dramatically reduced NPH I mediated transcription termination. Deletion of the terminal F(631), or substitution of F(631) with alanine, reduced binding to H4L and eliminated termination activity. These observations demonstrate that the terminal five amino acids directly participate in binding to RNA polymerase and in early gene transcription termination.