Aim: This study identified protein-protein interactions among the biosynthetic machinery responsible for exopolysaccharide (EPS) production in Streptococcus thermophilus MR-1C. Methods and Results: Protein-protein interactions were investigated using the yeast two-hybrid system. A strong protein-protein interaction was detected between the transmembrane activation protein Wzd and the protein tyrosine kinase Wze. Weaker protein-protein interactions were detected between two duplicate Wze proteins and between Wze and the phosphotyrosine phosphatase Wzh. Protein-protein interactions involving a Wzd/Wze fusion protein and Wzd and Wze may indicate that these proteins form multi-protein complexes. All combinations of the Wzh, Wzd, Wze, Wzg (regulation), CpsE (glycosyl-1-phosphate transferase), CpsS (polymerization), CpsL (unknown), CpsW (regulation) and CpsU (membrane translocation) were analysed for protein-protein interactions but no additional interactions were discovered using the yeast two-hybrid system. Conclusions: Interactions among the phosphotyrosine phosphatase, tyrosine kinase, and transmembrane activation protein are important in the regulation of capsule biosynthesis in Strep. thermophilus MR-1C. Significance and Impact of the Study: This study provides some valuable insight into the organization and interactions between the many proteins involved in EPS production. A better understanding of this process may facilitate the genetic manipulation of capsule production to impart desirable properties to dairy starter cultures.