||The hematopoietic-specific Gα16 and Gα14 link a variety of G protein-coupled receptors (GPCRs) to phospholipase Cβ (PLCβ) stimulation. Recent studies reveal that several Gα subunits are capable of activating signal transducer and activator of transcription (STAT) proteins. In the present study, we investigated the mechanism by which Gα16 and Gα14 mediate the stimulation of STAT3. In HEK 293 cells, constitutively active mutants of Gα16 (Gα16QL) and Gα14 (Gα14QL) stimulated STAT3-dependent luciferase activity as well as the phosphorylation of STAT3 at both Tyrosine705 and Serine727. Gα16QL- and Gα14QL-induced STAT3 activation was inhibited by U0126, a Raf-1 inhibitor, and coexpression of the dominant negative mutants of Ras and Rac1. Gα16 and Gα14 were capable of interacting with tetratricopeptide repeat 1 (TPR1) by coimmunoprecipitation. In particular, Gα16QL and Gα14QL appeared to be more tightly associated with TPR1. Inhibition of PLCβ, protein kinase C (PKC), and calmodulin-dependent kinase II (CaMKII) by their respective inhibitors also suppressed Gα16QL- and Gα14QL-induced STAT3 activation. The involvement of tyrosine kinases such as c-Src and Janus kinase 2 and 3 (JAK2 and JAK3) in Gα16QL- and Gα14QL-induced activation of STAT3 was illustrated by the combined use of selective inhibitors and dominant negative mutants, but no physical association between Gα14QL and c-Src could be detected by coimmunoprecipitation. PLCβ, PKC and CaMKII were shown to be involved in Gα14QL-mediated c-Src phosphorylation. c-Jun N-terminal kinase, p38 MAPK, RhoA, Cdc42 and phosphatidylinositol-3 kinase did not appear to be required for Gα16QL- and Gα14QL-induced STAT3 activation. Similar observations were obtained with human erythroleukemia (HEL) cells, where STAT3 phosphorylation was stimulated by C5a or DPDPE in a PTX-insensitive manner. Collectively, these results highlight the important regulatory roles of the PLCβ/CaMKII/PKC, Ras/Raf/MEK/ERK and c-Src/JAK pathways on the stimulation of STAT3 by activated Gα16 and Gα14. Demonstration of the involvement of different kinases in Gα16QL- and Gα14QL-induced STAT3 activation supports the involvement of multiple signaling pathways in the regulation of transcription by G proteins.