The optimal conditions for the endogenous phosphorylation of hen spinal cord cytosolic and membrane proteins with 5 µM [y-32P]ATP, 10 mM MgCl2, were determined by 10% SDS-polyacrylamide gel electrophoresis, autoradiography, and microdensitometry. Phosphate incorporation increased linearly with concentrations ranging from 35-75 µg/100 µl for cytosolic proteins and 21-125 µg/200 µl for membrane proteins. Optimal incubation times, temperatures, and pH values were 60 s, 30°C, and 6.0, respectively, for spinal cord cytosolic proteins and 15 s, 45°C, and 8.0, respectively, for spinal cord membranes. Prominent species differences in protein phosphorylation between these fractions in hens and similarly prepared fractions in rats, co-electrophoresed, include 80K and 30K protein phosphate acceptors unique to rat spinal cord cytosol, 60K and 16K protein phosphate acceptors characteristic of rat spinal cord membranes, a 50K protein phosphate acceptor present only in hen spinal cord membranes, and greater phosphorylation of a more abundant 20K protein in both hen spinal cord fractions. The functional significance of these differences is presently unclear. However, their characterization provides a basis from which to launch future investigations of the biochemistry, pharmacology, and toxicology of spinal cord protein phosphorylation and indicates that caution should be exercised in the choice of an animal model with characteristics appropriate to those of the system it is representing.