JAK3 is the only known protein tyrosine kinase associating with IL-2R gamma. This interaction is supposed to be very important to IL-2 signaling. In order to identify the critical residues for these two molecular interactions and the following signal events, various mutants of gammac and JAK3 were constructed on the basis of computer analysis. The direct interaction was determined via the yeast two-hybrid system, while the signaling was analyzed with reporter genes under the control of the c-fos, c-myc, or tnf-beta promoters, respectively. Results showed that there are two key sites on gammac involved in this interaction and the following signal transduction: the critical one is E327 via electrostatic interaction, the other is L293 via hydrophobic interaction. As to JAK3, the data indicated that Y100 is important for the interaction with gammac. These results also document that the requirement for interaction between gammac and JAK3 is different to activate different signaling pathways mediated by gammac, such as c-fos, c-myc, and JAK-STAT.