Protein-protein interactions mediated by the Src homology 3 (SH3) domain have been implicated in the regulation of receptor functions for subcellular localization of proteins and the reorganization of cytoskeleton. The experiments described in this article begin to identify the interaction of the SH3 domain of Src tyrosine kinase with the guanylyl cyclase C receptor after activation with Escherichia coli heat-stable enterotoxin (ST). Only one of two post-translationally modified forms of guanylyl cyclase C from T84 colonic carcinoma cells bind to GST-SH3 fusion protein of Src and Hck tyrosine kinases. Interestingly, the GST-Src-SH3 fusion protein showed 2-fold more affinity to native guanylyl cyclase C in solution than the GST-Hck-SH3 fusion protein. The affinity of the GST-Src-SH3 fusion protein to guanylyl cyclase C increased on desensitization of receptor in vivo. An in vitro cyclase assay in the presence of GST-Src-SH3 fusion protein indicated inhibition of the catalytic activity of guanylyl cyclase C. The catalytic domain recombinant protein (GST-GCD) of guanylyl cyclase C could pull-down a 60-kDa protein that reacted with Src tyrosine antibody and also showed autophosphorylation. These data suggest that SH3 domain-mediated protein-protein interaction with the catalytic domain of guanylyl cyclase C inhibited the cyclase activity and that such an interaction, possibly mediated by Src tyrosine kinase or additional proteins, might be pivotal for the desensitization phenomenon of the guanylyl cyclase C receptor.