Plasmodesmata are channels that bridge the cell walls of plant cells, allowing regulated transport of molecules between neighbouring cells. We have used a proteomic strategy to identify putative plasmodesmata-associated proteins in the giant-celled green alga Chara corallina. Proteins were extracted from the plasmodesmata-rich nodal complexes and the middle of the long internodal cells, which do not contain plasmodesmata. Comparison of protein spot patterns generated by two-dimensional gel electrophoresis of both the soluble and cell wall fractions from the two cell types was done. Fifty-eight spots that were common to the nodal and internodal soluble fractions were analysed by matrix assisted laser desorption/ionisation-time of flight mass spectrometry, and peptide mass fingerprint data were used to search the database. Matches were made to four of these spots, in each case to housekeeping proteins. Further, a number of nodal specific spots were identified, 11 from the soluble fraction and nine from the wall fraction. These spots were excised from the gels and analysed by liquid chromatography tandem mass spectrometry to obtain peptide sequence. Database searches suggest that these spots include homologues to previously identified plasmodesmata-associated proteins cp-wap13 and heat shock cognate 70, as well as RNA-binding proteins, eukaryotic initiation factor 4A and a beta-1,3-glucanase. Several spots remained unidentified providing exciting new candidate plasmodesmata-associated proteins.