Aglaothamnion neglectum Feldman-Mazoyer has two γ subunits, γ31 and γ33, that are associated with phycoerythrin in the light-harvesting phycobilisomes. We demonstrate that these subunits are spatially separated within the phycobilisome, with the γ31 subunit present at the distal end of phycobilisome rods and the γ33 subunit present on the proximal end. These subunits are thought to link phycoerythrin hexamers together in the rod substructure, serving a role analogous to that of linker polypeptides of cyanobacteria (although unlike the cyanobacterial linker polypeptides they are chromophorylated). The sequencing of tryptic polypeptides of the γ subunits enabled us to prepare oligonucleotides encoding different regions of γ31. These oligonucleotides were used as primers to generate a probe for isolating a γ31 cDNA clone. Characterization of the cDNA clone predicts a polypeptide of 280 amino acids with a 42 amino acid presequence that is characteristic of a transit peptide, the peptide that targets proteins to chloroplasts of vascular plants. The γ31 subunit has 50% similarity to the previously characterized γ33 subunit but has no identifiable similarity to functionally related polypeptides present in cyanobacterial phycobilisomes or to any other polypeptides in the databases. A repeat of 95 amino acids is present in the red algal γ subunit sequences, suggesting that these proteins were generated by a gene duplication followed by fusion of the duplicate sequences.