hGrb10alpha (previously named Grb-IR) is a Src-homology 2 domain-containing protein that binds with high affinity to the tyrosine-phosphorylated insulin receptor and insulin-like growth factor-1 receptor. At least two isoforms of human Grb10, (hGrb10alpha and hGrb10beta), which differ in the pleckstrin homology (PH) domain and the N-terminal sequence, have previously been identified in insulin target tissues such as human skeletal muscle and fat cells. Here we report the cloning of the third isoform of the hGrb10 family (hGrb10gamma) from human skeletal muscle and its localization to human chromosome 7. We have also determined the human chromosome localization of Grb7 to 17q21-q22 and Grb14 to chromosome 2. hGrb10gamma contains an intact PH domain and an N-terminal sequence that is present in hGrb10alpha but absent in hGrb10beta. RNase protection assays and Western blot analysis showed that hGrb10alpha and hGrb10gamma are differentially expressed in insulin target cells including skeletal muscle, liver, and adipocyte cells. hGrb10gamma is also expressed in HeLa cells and various breast cancer cell lines. The protein bound with high affinity to the insulin receptor in cells, and the interaction was dependent on the tyrosine phosphorylation of the receptor. hGrb10gamma also underwent insulin-stimulated membrane translocation and serine phosphorylation. hGrb10gamma phosphorylation was inhibited by PD98059, a specific inhibitor of mitogen-activated protein kinase kinase, and wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase. Taken together, our data suggest that hGrb10 isoforms are potential downstream signaling components of the insulin receptor tyrosine kinase and that the PH domain may play an important role in the involvement of these isoforms in signal transduction pathways initiated by insulin and other growth factors.