Peripherin/rds (p/rds), an integral membrane protein from the transmembrane 4 (TMF4) superfamily, possesses a multi-functional C-terminal domain that plays crucial roles in rod outer segment (ROS) disk renewal and structure. Here, we report that the calcium binding protein calmodulin (CaM) binds to the C-terminal domain of p/rds. Fluorescence spectroscopy reveals Ca 2+ -dependent association of CaM with a polypeptide corresponding to the C-terminal domain of p/rds. The fluorescence anisotropy of the polypeptide upon CaM titration yields a dissociation constant (K D ) of 320 ± 150 nM. The results of the fluorescence experiments were confirmed by GST-pull down analyses in which a GST-p/rds C-terminal domain fusion protein was shown to pull down CaM in a calcium-dependent manner. Moreover, molecular modeling and sequence predictions suggest that the CaM binding domain resides in a p/rds functional hot spot, between residues E314 and G329. Predictions were confirmed by peptide competition studies and a GSTp/rds C-terminal domain construct in which the putative Ca 2+ /CaM binding site was scrambled. This GST-polypeptide did not associate with Ca 2+ /CaM. This putative calmodulin domain is highly conserved between human, mouse, rat, and bovine p/rds. Finally, the binding of Ca 2+ /CaM inhibited fusion between ROS disk and ROS plasma membranes as well as p/rds C-terminaldomain-induced fusion in model membrane studies. These results offer a new mechanism for the modulation of p/rds function.Photoreceptor peripherin/rds (p/rds 1 ) and the non-glycosylated homologue ROM-1 belong to a growing and increasingly complex family of proteins, the tetraspanins (also known as the TMF4 (transmembrane 4 superfamily)) (1, 2). P/rds shares 41% sequence homology