Mycoplasma hyopneumoniae causes swine enzootic pneumonia, an important disease in the swine industry. Adherence of M. hyopneumoniae to the cilia of the tracheal epithelial cells is required to establish infection. Previous studies have identified a 97-kDa protein (P97) as the putative ciliary adhesin. To further characterize the P97 protein, the gene has been cloned, its DNA sequence analyzed, and the function of the P97 protein expressed in Escherichia coli studied. These results demonstrated that recombinant P97 has swine ciliary adherence activity. Further analysis revealed that the P97 gene was translated as a 124.9-kilodalton (kDa) protein and subjected to proteolytic cleavage at amino acid 195, resulting in a protein with a predicted molecular weight of 102 kDa. The translated P97 protein showed a high degree of hydrophilicity and contained no cysteine residues or acylation sites, confirming previous findings that P97 was not an integral membrane protein. Two repeat sequences, R1 and R2, were identified at the carboxy end of P97. The R1 sequence contained 15 repeats of AAKP(V/E) and was shown to function as the P97 ciliary binding motif. The adherence-blocking monoclonal antibody F1B6 antigenic epitope was also mapped to the 5' end of the R1 sequence. Analysis of different M. hyopneumoniae isolates displaying variation in cilia binding activity, showed that the variation was not due to expression of P97, the number of R1 copies, or changes in the AAKP(V/E) repeat sequence. Thus, another mechanism must be functioning to control cilium binding activity in M. hyopneumoniae. The R2 sequence contained four repeats of NQGKK(S/A)EG(A/T)P and showed a high degree of homology to ribosome binding proteins. Analysis of the P97 contig region showed that the P97 gene was the first of a two gene operon, designated the P97 operon. The second open reading frame coded for a possible membrane protein with a predicted molecular weight of 102 kDa. It had no sequence homology with any known sequence. Hybridization studies showed that the P97 operon sequence existed as multiple copies in the M. hyopneumoniae chromosome. These findings support the hypothesis that P97 is involved in ciliary adherence of M. hyopneumoniae.