Subtypes of the calcium-independent receptors for alpha-latrotoxin (CIRL1-3) define a distinct subgroup within the large family of the seven-transmembrane region cell surface receptors. The physiological function of CIRLs is unknown because neither extracellular ligands nor intracellular coupling proteins (G-proteins) have been identified. Using yeast two-hybrid screening, we identified a novel interaction between the C termini of CIRL1 and -2 and the PSD-95/discs large/ZO-1 (PDZ) domain of a recently discovered multidomain protein family (ProSAP/SSTRIP/Shank) present in human and rat brain. In vitro, CIRL1 and CIRL2 interacted strongly with the PDZ domain of ProSAP1. The specificity of this interaction has been verified by in vivo experiments using solubilized rat brain membrane fractions and ProSAP1 antibodies; only CIRL1, but not CIRL2, was co-immunoprecipitated with ProSAP1. In situ hybridization revealed that ProSAP1 and CIRL1 are co-expressed in the cortex, hippocampus, and cerebellum. Colocalization was also observed at the subcellular level, as both CIRL1 and ProSAP1 are enriched in the postsynaptic density fraction from rat brain. Expression of all three CIRL isoforms is highly regulated during postnatal brain development, with CIRL3 exhibiting its highest expression levels immediately after birth, followed by CIRL2 and finally CIRL1 in aged rats.