Peripherin/rds (P/rds) is a disk rim protein that assembles into homo and hetero complexes with its nonglycosylated homologue, Rom-1, to maintain the integrity of the photoreceptor outer segment. Mutations in the rds gene have been identified in a variety of human retinal degenerative diseases. More than 70% of these mutations are located in the second intradiscal (D2) loop, highlighting the functional importance of this region. This study examines the involvement of different regions of the D2 loop in protein associations using a GST pull-down assay and a heterologous coexpression system. The pull-down assay suggests an association of the N-terminal portion (Phe(120)-Phe(187)) of the D2 loop with Rom-1 as well as with other P/rds molecules. Through peptide competition experiments, the region between Cys(165) and Asn(182) of the D2 loop has been identified as the domain for these associations. In a COS-1 cell heterologous expression system, coexpression of the D2 loop along with the intact P/rds and Rom-1 hindered the association of the two full-length proteins. In contrast to the homo association of P/rds molecules, it seems that the hetero association of P/rds with Rom-1 has a more stringent structural requirement. This work defines the crucial domain of the D2 loop, which mediates homo and hetero associations, specifically the regions that lay between Cys(165) and Asn(182). Elucidation of the molecular mechanisms behind the protein-protein associations of P/rds and its partners may reveal the pathogenic defects arising from the most common mutations in this gene.