The gene coding for the large subunit of herpes simplex virus type 2 ribonucleotide reductase (RR) (ICP10) has a unique 5′ terminal domain the product of which has a serine/threonine (Ser/Thr) protein kinase (PK) catalytic domain preceded by a transmembrane (TM) segment. Because ICP10 localizes on the cell surface and is internalized by the endocytic pathway like an activated growth factor receptor (Hunteret al.,1995,Virology210, 345–360), we asked whether it is ligand-inducible in order to examine whether it has intrinsic transphosphorylating activity. We constructed a chimeric expression vector that contains the extracellular and TM domains of the epidermal growth factor receptor (EGFR) joined to the intracellular PK and RR domains of ICP10 (pCH5) and established constitutively expressing cell lines in NIH3T3 2.2 cells that do not express EGFR. The chimeric protein, designated p210 CH5 , localized to the surface of these cells as determined by immunofluorescent staining with MAb EGFR, and it bound 125 I-EGF. p210 CH5 coprecipitated with protein species p170, p120, p88, p60, p44, p34, and p25. EGF treatment activated the PK activity of p210 CH5 , resulting in its autophosphorylation and the phosphorylation of the p120, p88, and p34 species. Immunoprecipitation/immunoblotting with anti-ras-GAP antibody and phosphoamino acid analysis indicated that p120 isras-GAP and it is phosphorylated on Ser/Thr residues. The identities of the phosphorylated p88 and p34 are still unknown. The data indicate that when fused to a ligand-regulated extracellular domain (EGFR), the ICP10 PK auto- and transphosphorylating activities are ligand-inducible. These findings support the interpretation that the ICP10 PK activity is intrinsic and indicate thatras-GAP is one of its phosphorylation substrates.