The attachment of virulent Mycoplasma pneumoniae to the ciliated epithelium of the respiratory tract involves a surface protein designated P1. Our previous determination of the nucleotide sequence of the P1 attachment-protein gene revealed that it is flanked by open reading frames (ORFs) and there is no obvious ribosome-binding site (RBS) or transcription termination sequence in the adjacent regions. We extended this analysis by cloning and sequencing the 18-kb region containing the P1 gene. This study indicates that the P1 gene is transcribed as part of a larger polycistronic message. The P1 operon is composed of the P1 gene and two predicted genes, designated ORF-4 and ORF-6. The gene order is ORF-4, P1, ORF-6 with intervening regions of 12 and 5 nt, respectively. ORF-4 and ORF-6 have respective coding capacities for proteins of Mr approximately equal to 28,000 and Mr approximately equal to 130,000. Putative promoter and RBS sequences which correspond closely to those found in Escherichia coli and Bacillus subtilis, as well as a sequence indicative of a transcription terminator, have been found in the flanking sequences. The transcription start point has been determined by primer extension of M. pneumoniae RNA.