Detailed analysis of the site-specific phosphorylation of a glucan phosphate from Saccharomyces cerevesiae has been carried out by 2D NMR techniques. Phosphorylation has been shown to be limited to the C-6 and C-2 positions, with the C-6 resonance showing two slightly different environments. Phosphorylation at C-4 is hindered due to proximity effects with the neighboring glucose ring oxygen. Noncovalently bound, nondialyzable phosphate appears to be coordinated to the nonphosphorylated HO-2 group of the helical polymer.