Rim1, a brain-specific Rab3a-binding protein, localizes to the presynaptic cytomatrix, and plays an important role in synaptic transmission and synaptic plasticity. Rim2, a homologous protein, is more ubiquitously expressed, and is found in neuroendocrine cells as well as in brain. Both Rim1 and Rim2 contain multiple domains, including an N-terminal Zn-finger, which in Rim1 strongly enhances secretion in chromaffin and PC12 cells. The yeast two-hybrid technique identified 14-3-3 proteins as ligands of the N-terminal domain. In vitro protein binding experiments confirmed a high affinity interaction between the N-terminus of Rim1 and 14-3-3. The N-terminal domain of Rim2 also bound 14-3-3. The binding domains were localized to a short segment just C-terminal to the Zn finger. 14-3-3 proteins bind to specific phosphoserine residues. Alkaline phosphatase treatment of N-terminal domains of Rim1 and Rim2 almost completely inhibited the binding of 14-3-3. Two serine residues in Rim1 (S241 and S287) and one serine residue in Rim2 (S335) were required for 14-3-3 binding. Incubation with CaMKII greatly stimulated the interaction of recombinant N-terminal Rim but not the S241/287A mutant with 14-3-3, again indicating the importance of the phosphorylation of these residues for the binding. Rabphilin3, another Rab3a effector, also bound 14-3-3. Serine to alanine mutations identified S274 as the likely phosphorylated residue to which 14-3-3 binds. Because the phosphorylation of this residue had been shown to be stimulated upon depolarization in brain slices, the interaction of 14-3-3 with Rabphilin3 may be important in the dynamic function of CNS neurons.