RIM-binding proteins (RIM-BPs) were identified as binding partners of the presynaptic active zone proteins RIMs as well as for voltage-gated Ca(2+)-channels. They were suggested to form a functional link between the synaptic-vesicle fusion apparatus and Ca(2+)-channels. Here we show that the RIM-BP gene family diversified in different stages during evolution, but retained their unique domain structure. While invertebrate genomes contain one, and vertebrates include at least two RIM-BPs, we identified an additional gene, RIM-BP3, which is exclusively expressed in mammals. RIM-BP3 is encoded by a single exon of which three copies are present in the human genome. All RIM-BP genes encode proteins with three SH3-domains and two to three fibronectin III repeats. The flanking regions diverge in size and sequence and are alternatively spliced in RIM-BP1 and -2. Quantitative real-time RT-PCR and in situ hybridization analyses revealed overlapping but distinct expression patterns throughout the brain for RIM-BP1 and -2, while RIM-BP3 was detected at high levels outside the nervous system. The modular domain structure of RIM-BPs, their expression pattern and the conservative expansion during evolution shown here support their potential role as important molecular adaptors.