Sidekick-1, a cell adhesion molecule of the immunoglobulin superfamily, is up-regulated in glomerular podocytes in the collapsing glomerulopathy of HIV-associated nephropathy (HIVAN). Sidekick-1 and its ortholog sidekick-2 have also been shown to function as neuronal targeting molecules, guiding developing neurons to specific synapses. In the current work, we overexpress mouse sidekick-1 and -2 in HEK 293 T cells in order to characterize their binding specificities. Cells transiently transfected with either sidekick-1 or -2 cDNA formed separate aggregates when mixed together, demonstrating that sidekicks are homophilic adhesion molecules. The transfection of the short splice variant (lacking the first two Ig domains) or a construct encoding sidekick-1 with the second Ig domain deleted both resulted in nearly abolished adhesion. A beta-sheet strand peptide containing the sequence QLVILA corresponding to an amino acid sequence in the second Ig domain of sidekick-1 showed specific interaction with the recombinant first Ig domain-His protein of sidekick-1. Cells expressing a mutant sidekick-1 where the binding sequence QLVILA is deleted failed to mediate significant adhesion. Furthermore, cells transfected with a chimeric sidekick, where the first two Ig domains of sidekick-2 are replaced with the corresponding two Ig domains of sidekick-1, form aggregates with sidekick-1-transfected cells. The reverse chimera, where the first two Ig domains of sidekick-2 are substituted onto sidekick-1, was similarly able to form aggregates with sidekick-2-transfected cells. These results establish that the first and second Ig domains of sidekick-1 and -2 are necessary and sufficient to mediate and target homophilic adhesion, and the QLVILA sequence is critical to the interaction. Understanding these functional domains has widespread implications in normal development and HIVAN pathogenesis.