Streptococcus gordonii M5 and DL1 each express two related adhesin polypeptides, SspA and SspB, which are members of the antigen I/II family of streptococcal surface proteins. The sspA and sspB genes are tandemly arranged in both strains, with sspA residing upstream of sspB. The genes are separated by approximately 400 nucleotides in S. gordonii DL1 and 1300 nucleotides in S. gordonii M5. The nucleotide sequence of the sspA/sspB intergenic region of strain M5 is reported and the difference in length compared to S. gordonii DL1 shown to arise from the presence of an insertion sequence, designated ISSg1, consisting of 1197 bp. The nucleotide sequence of ISSg1 is highly homologous to IS1167 to Streptococcus pneumoniae and is related to a lesser extent to other members of the IS1096 family of bacterial insertion sequences. It contains a single ORF of 1026 bp, encoding a putative transposase polypeptide of 342 amino acids. The deduced transposase sequence exhibits 93% identity with the transposase polypeptides encoded by IS1167. However, the S. gordonii protein lacks a 90 residue central domain that is present in the IS1167 transposase and in the transposase polypeptides encoded by the related IS elements. In addition, the organization of the inverted repeats flanking the transposase gene in S. gordonii differs from IS1167. Extension products generated from a sspB-specific primer indicated that transcription initiates within the intergenic region in both S-gordonii strains, suggesting that sspA and sspB are independently transcribed. Transcription appears to initiate 42 bases upstream of sspB in S. gordonii DL1. In contrast, sspB transcription in M5 initiates at least 125 bases upstream of sspB, in close proximity to the terminal inverted repeat of ISsg1. These results indicate that the sspB promoter of S. gordonii M5 and DL1 are not conserved and suggest that ISSg1 sequences may play a role in directing the expression of sspB in S. gordonii M5.