The effect of a single 750-mg/kg oral dose of tri-o-cresyl phosphate (TOCP) on the endogenous phosphorylation of brain microtubule preparations and spinal cord neurofilaments was assessed in hens after the development of delayed neurotoxicity. Protein phosphorylation with [gamma-32P]ATP was analyzed by one-dimensional and two-dimensional gel electrophoresis, autoradiography, and microdensitometry. TOCP treatment enhanced the Ca2+- and calmodulin-dependent phosphorylation of tubulin in crude chicken brain cytosol (160% for alpha-tubulin and 140% for beta-tubulin) and cold-stable microtubules (165% and 155% for alpha- and beta-tubulin, respectively). Microtubule-associated protein 2 (MAP-2) phosphorylation was also increased in brain fractions studied--i.e., brain cytosol (145%), cold-stable microtubules (133%), and cold-labile microtubules (328%). There was significant increase in phosphorylation of a 70-kDa protein in the brain cytosol and in the cold-stable microtubule fractions. TOCP also stimulated the phosphorylation of spinal cord proteins of 70 kDa (119%) and 160 kDa (129%) in a Mg2+-dependent manner. Addition of Ca2+ and calmodulin further enhanced the phosphorylation of these 70-kDa (563%) and 160-kDa (221%) proteins as well as of 52-, 59-, and 210-kDa proteins by as much as 126%, 160%, and 196%, respectively. Two-dimensional electrophoresis was carried out to identify these proteins. They were confirmed as alpha- and beta-tubulin (52 and 59 kDa) in brain and spinal cord preparations and the neurofilament triplet proteins (70, 160, and 210 kDa) in the spinal cord preparation. The 70-kDa protein in brain was not neurofilament in origin. Peptide mapping using Staphylococcus aureus V8 protease showed the brain and spinal cord cytoskeletal proteins have identical phosphopeptide patterns in control and TOCP-treated hens, indicating that it was unlikely that the phosphorylation sites were altered by TOCP treatment.