Voltage-gated Na+ channel (VGSC) β1 and β2 subunits are multifunctional, serving as both channel modulators and cell adhesion molecules (CAMs). The purpose of this study was to determine whether VGSC β3 subunits function as CAMs. The β3 extracellular domain is highly homologous to β1, suggesting that β3 may also be a functional CAM. We investigated the trans homophilic cell adhesive properties of β3, its association with the β1-interacting CAM contactin, as well as its ability to interact with the cytoskeletal protein ankyrin. Our results demonstrate that, unlike β1, β3 does not participate in trans homophilic cell-cell adhesion or associate with contactin. Further, β3 does not associate with ankyrinG in a heterologous system. Previous studies have shown that β3 interacts with the CAM neurofascin-186 but not with VGSC β1. Taken together, these findings suggest that, although β1 and β3 exhibit similar channel modulatory properties in heterologous systems, these subunits differ with regard to their homophilic and heterophilic CAM binding profiles. © 2009 Elsevier Ireland Ltd. All rights reserved.