Taxilin is novel binding partner of the syntaxin family, which is implicated in intracellular vesicle traffic. However, precise binding properties of taxilin to the syntaxin family remain to be clarified. Then, we here further investigated the interaction of taxilin with the syntaxin family by use of recombinant taxilins. Syntaxin-1a, -3, and -4 bound to taxilin in a dose-dependent and saturable manners. The concentrations of syntaxin-1a, -3, and -4 giving a half-maximal binding to taxilin were about 1.5, 3.0, and 1.0microM, respectively. The interaction of taxilin with syntaxin-1a was inhibited by SNAP-25 or Munc18 in a dose-dependent manner. When recombinant taxilin was incubated with the extract from the rat brain crude membrane fraction, recombinant taxilin bound to syntaxin-1s free of at least VAMP2, SNAP-25, and Munc18. These results suggest that taxilin interacts with the syntaxin family which does not form at least the SNARE complex.