SspA and SspB (antigen I/II family proteins) can bind Streptococcus gordonii to other oral bacteria and also to salivary agglutinin glycoprotein, a constituent of the salivary film or pellicle that coats the tooth. To learn if SspA and SspB are essential for adhesion and initial biofilm formation on teeth, S. gordonii DL1 was incubated with saliva-coated hydroxyapatite (sHA) for 2 h in Todd-Hewitt broth with 20% saliva to develop initial biofilms. Sessile cells attached to sHA, surrounding planktonic cells, and free-growing cells were recovered separately. Free-growing cells expressed more sspA-specific mRNA and sspB-specific mRNA than sessile cells. Free-growing cells expressed the same levels of sspA and sspB as planktonic cells. Surprisingly, an SspA- SspB- mutant strain showed 2.2-fold greater biofilm formation on sHA than wild-type 5. gordonii DL1. To explain this observation, we tested the hypothesis that inactivation of sspA and sspB genes altered the expression of other adhesin genes during initial biofilm formation in vitro. When compared to wild-type cells, expression of scaA and abpB was significantly up-regulated in the SspA- SspB- strain in sessile, planktonic, and free-growing cells. Consistent with this finding, ScaA antigen was also overexpressed in planktonic and free-growing SspA- SspB- cells compared to the wild type. SspA/B adhesins, therefore, were strongly suggested to be involved in the regulation of multiple adhesin genes.