The regulation and substrate specificity of microglial phosphotyrosine (ptyr) increases accompanying motor neuron degeneration in the rat spinal cord induced by injection of the cytotoxic lectin, ricin, into sciatic nerve were examined using specific enzyme inhibitors, immunohistochemistry, and Western blot analyses. Optical density measurements of immunostained sections show that microglial ptyr levels are elevated at 3 days postinjection. This period coincides with initial stages of neuronal degeneration, and ptyr levels are maximal at 7 days. We next asked whether this increase is due to increased tyrosine kinase or decreased tyrosine phosphatase activities by assaying ptyr immunostaining in animals that received osmotic pump infusion of the nonreceptor tyrosine kinase inhibitor, herbimycin A, for the 7-day survival period. When compared to the control ventral horn, microglial ptyr on the experimental side was attenuated by at least 45% in the presence of herbimycin A. In order to identify microglial substrates undergoing increased tyrosine phosphorylation, Western blot analysis was performed on hemicord and punch biopsy samples from control and experimental sides following ricin injection. A subset of two proteins was identified whose increased ptyr was almost completely attenuated in the herbimycin-A-treated animals. We conclude that the data support earlier indications that upregulation of microglial tyrosine phosphorylation is a key early event in response to neuronal injury. Further, this upregulation is due to turning on tyrosine kinase activities, particularly nonreceptor kinases, and the end product is phosphorylation of a very limited number of substrates. This suggests the activation of specific tyrosine phosphorylation pathways, which may represent critical therapeutic intervention points, rather than a global response. The results are discussed in terms of recent cell culture models of microglial activation and earlier data demonstrating elevated microglial ptyr in neurodegenerative disease.