The residue proposed to serve as the catalytic base for phosphoryl transfer, Asp-813, of the human epidermal growth factor receptor (EGFR) was mutated to Ala, and the mutant receptor (D813A) was expressed in Chinese hamster ovary (CHO) cells. Partially purified D813A exhibited no detectable kinase activity in the absence or presence of EGF. A low level of EGF-stimulable phosphorylation of D813A was detectable in intact cells, apparently due to the activity of an associated Tyr kinase(s). As previously observed for kinase-inactive Lys-721 mutants, EGF binding to D813A stimulates mitogen-activated protein kinase activity. Surprisingly, and unlike results reported for Lys-721 mutants, D813A is capable of stimulating both 86Rb+ uptake and DNA synthesis in response to EGF. These data suggest not only that Asp-813 is critical to the catalytic activity of the EGFR but also that differences may exist in the signaling properties of kinase-negative Lys-721 and kinase-negative Asp-813 EGFR mutants.