Using S1 nuclease assays, we located the sites of initiation of transcription of the beta-lactamase gene on Tn1 and Tn2. Transcription in Tn2, like that in Tn3, occurred from the P3 promoter, whereas transcription in Tn1 was initiated by two stronger and overlapping promoters, Pa and Pb. The nucleotide sequences of Tn1 and Tn2 were determined over a 1,195-base-pair segment constituting most of the sequences of the tnpR and bla genes and the intervening region. There were six base-pair differences between Tn1 and Tn3. One in the bla regulatory region accounted for the presence of the Pa and Pb promoters, and another in the bla structural gene is consistent with the isoelectric focusing difference found between the Tn1 and Tn3 enzymes. In contrast, there were 24 base-pair differences between Tn2 and Tn3, most of them clustered in one segment of the tnpR gene.