The structure–function relationship of proteinaceous filaments in sieve elements has long been a source of investigation in order to understand their role in the biology of the phloem. Two phloem filament proteins AtSEOR1 (At3g01680.1) and AtSEOR2 (At3g01670.1) in Arabidopsis have been identified that are required for filament formation. Immunolocalization experiments using a phloem filament-specific monoclonal antibody in the respective T-DNA insertion mutants provided an initial indication that both proteins are necessary to form phloem filaments. To investigate the relationship between these two proteins further, green fluorescent protein (GFP)–AtSEO fusion proteins were expressed in Columbia wild-type and T-DNA insertion mutants. Analysis of these mutants by confocal microscopy confirmed that phloem filaments could only be detected in the presence of both proteins, indicating that despite significant sequence homology the proteins are not functionally redundant. Individual phloem filament protein subunits of AtSEOR1 and AtSEOR2 were capable of forming homodimers, but not heterodimers in a yeast two-hybrid system. The absence of phloem filaments in phloem sieve elements did not result in gross alterations of plant phenotype or affect basal resistance to green peach aphid (Myzus persicae).