The goal of the present study was to begin characterizing the amylase-binding component(s) on the surface of Streptococcus gordonii G9B. Alkali extracts but not phenol-water extracts of this bacterium inhibited 125I-amylase binding to S. gordonii G9B. To identify the bacterial components involved in amylase binding, the alkali extract was subjected to affinity chromatography on amylase-Sepharose. Immunoblotting with a rabbit antiserum against S. gordonii G9B revealed that a 20-kDa streptococcal component was eluted from the amylase-Sepharose with 1% sodium dodecyl sulfate (SDS), 2 M KSCN, or 0.1 M sodium citrate buffer, pH 4.5. Subsequently, the 20-kDa component was prepared from alkali extracts by electroelution from preparative SDS electrophoresis or by gel filtration chromatography. This component was trypsin sensitive, and an antibody raised against it inhibited the binding of 125I-amylase to S. gordonii G9B. Indirect immunofluorescence microscopy and immunogold electron microscopy demonstrated that both bound amylase and the 20-kDa component were localized to the cell division septum on dividing cells or to polar zones on single cells. In addition, exponentially growing bacteria bound more 125I-amylase than stationary-phase cells did. Collectively, these results suggest that a 20-kDa amylase-binding component is present on the surface of the nascent streptococcal cell wall.