We identified a novel gene of bacteriophage T4, gene 61.5, which appears to be involved in protein synthesis late in infection. Northern blot analysis revealed that a mutant of 61.5 accumulated truncated transcripts of representative late genes. Using a double mutant of genes 61.5 and 55, which prevents transcription of late genes, we demonstrate that even transcripts of middle genes, while full-length when initially expressed, are similarly truncated at later stages of infection. These results indicate that the abnormality in transcript length occurs late in infection, regardless of whether the transcript derives from a middle or a late gene. Primer-extension analysis revealed that the 5' ends of the late gene 23 transcripts that accumulated in gene 61.5 mutant-infected cells were located at internal discrete sites as well as at the expected transcription start site. Moreover, the decay rates of full-length transcripts from genes uvsY or 45 were more than twofold faster in the absence of a functional gene 61.5. These results suggest that mutation of gene 61.5 activates endonucleolytic cleavage of middle and late transcripts, probably by RNase M.