After T4 bacteriophage infects Escherichia coli, a peptide tau, produced under the control of a phage gene, binds to the host valyl transfer ribonucleic acid synthetase (EC 6.1.1.9) and thereby changes several of its physicochemical properties. The interaction of tau with the host enzyme was investigated in vitro after extensively purifying the factor from T4-infected E. coli using a rapid purification procedure. The tau preparation migrated as a single, protein-staining band with a molecular weight of 11,000 during sodium dodecyl sulfate-gel electrophoresis. The purified peptide completely converted partially purified valyl-tRNA synthetase from uninfected E. coli into the form present in cell-free extracts prepared from virus-infected bacteria. The enzyme modified in vitro also exhibited the enhanced affinity for tRNA characteristic of the viral form of valyl-tRNA synthetase. The addition of bulk tRNA from E. coli B, tRNAVal, or tRNA1Val to enzyme modified in vitro increased its sedimentation rate to that of enzyme prepared from phage-infected cells. Amino acid analysis of the purified tau peptide revealed a relatively high concentration of the amino acids lysine and alanine, and a lack of detectable proline, tyrosine, phenylalanine, and methionine.