Background: G protein-coupled receptors constitute the largest family of cell surface receptors in the mammalian genome. As the core of the G protein signal transduction machinery, the Gα subunits are required to interact with multiple partners. The GTP-bound active state of many Gα subunits can bind a multitude of effectors and regulatory proteins. Yet it remains unclear if the different proteins utilize distinct or common structural motifs on the Gα subunit for binding. Using Gα16 as a model, we asked if its recently discovered adaptor protein tetratricopeptide repeat 1 (TPR1) binds to the same region as its canonical effector, phospholipase Cβ (PLCβ). Results: We have examined the specificity of Gα16/TPR1 association by testing a series of chimeras between Gα16 and Gαz. TPR1 co-immunoprecipitated with Gα16 and more tightly with its constitutively active Gα16 QL, but not Gαz. Progressive replacement of Gα16 sequence with the corresponding residues of Gαz eventually identified a stretch of six amino acids in the β3 region of Gα16 which are responsible for TPR1 interaction and the subsequent Ras activation. Insertion of these six residues into Gαz allowed productive TPR1-interaction. Since the β3 region only minimally contributes to interact with PLCβ, several chimeras exhibited differential abilities to stimulate PLCβ and Ras. The ability of the chimeras to activate downstream transcription factors such as signal transducer and activator of transcription 3 and nuclear factor κB appeared to be associated with PLCβ signaling. Conclusions: Our results suggest that Gα16 can signal through TPR1/Ras and PLCβ simultaneously and independently. The β3 region of Gα16 is essential for interaction with TPR1 and the subsequent activation of Ras, but has relatively minor influence on the PLCβ interaction. Gα16 may utilize different structural domains to bind TPR1 and PLCβ. Conclusions: Our results suggest that G alpha(16) can signal through TPR1/Ras and PLC beta simultaneously and independently. The beta 3 region of G alpha(16) is essential for interaction with TPR1 and the subsequent activation of Ras, but has relatively minor influence on the PLC beta interaction. G alpha(16) may utilize different structural domains to bind TPR1 and PLC beta.