Bacteriophage lambda transcripts were synthesized in vitro using lambda b2 DNA and Escherichia coli RNA polymerase. RNA molecules initiating with ATP were labeled exclusively at the 5'-end by transcribing in the presence of [gamma-32P]ATP. They were analyzed by electrophoresis in 3.5% polyacrylamide, 7.5 M urea gels followed by autoradiography. The previously known 6 S rho-independent transcript and rho-dependent 9 S and 8 S transcripts were observed. A new rho-dependent 5 S transcript was also detected. The 5 S RNA was not the result of cleavage of larger RNAs by ribonuclease III. Analysis of partial ribonuclease T1 digestion products of isolated 5'-end-labeled transcripts showed that the 5 S RNA is synthesized from promoter pR, the promoter for the 9 S and 8 S RNA species. A similar analysis of transcripts labeled with 32P exclusively at the 3'-terminus by the post-transcriptional addition of a [32P]pCp moiety with T4 RNA ligase revealed the positions of guanosine nucleotides proximal to the 3'-hydroxyl end. This information, and inspection of the known DNA nucleotide sequence downstream from pr, allowed us to conclude that the 5 S RNA is a mixture of molecules 112 and 113 nucleotides long terminating in the middle of gene cro. The nucleotide sequence at the 3'-end of the 5 S RNA is similar to that of other rho-dependent transcripts and lacks the oligo(U) found at the terminus of rho-independent transcripts.