The expression of the T4 rII genes in uninfected cells has been examined by use of recombinant plasmids. Hybridization analysis of pulse-labelled RNA prepared from cells carrying pTB101, a plasmid that contains the end of T4 gene 60 and the beginning of gene rIIA, shows that about 0.7% of the labelled RNA is rII specific. By contrast, only 0.02% of pulse-labelled RNA prepared from cells carrying plasmid pTB301, which probably contains the middle-mode rIB promoter, may be rII specific. When separated strands of T4 DNA were used for hybridization, we found that the pTB101 transcripts have a strand specificity identical to that of the rIIA transcripts made during phage infection. The same strand specificity was observed irrespective of the orientation of the inserted DNA in the vector. This result argues that the transcripts initiate within the inserted DNA rather than somewhere else on the plasmid. We also found that essentially none of the pulse-labelled pTB101 RNA would hybridize to the DNA of a T4 deletion mutant that lacks the rIIA gene. This suggests that little of the gene 60 DNA of the plasmid is being transcribed. In addition to the rII transcript, a new protein of 56,000 Daltons molecular weight is found in cells carrying pTB101. Fingerprint analysis of the protein shows that it is specified by the rIIA gene of the plasmid. Taken together, these results indicate that transcription of the plasmid rIIA gene initiates at or near the beginning of the gene.