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This chapter discusses the solid-phase sequencing of ³²P-labeled phosphopeptides at picomole and subpicomole levels. Protein phosphorylation is usually restricted to the hydroxyamino acids, serine, threonine, and tyrosine, although phosphorylation of histidine can occur in some systems. Specificity determinants for site-specific phosphorylation are usually provided by structural signals located within amino acid sequences adjacent to the phosphorylation site. Because phosphorylation sites cannot be predicted with certainty from primary structure alone, there is a need for methodology to determine the sites experimentally. Indirect detection methods for the assignment of such phosphorylation sites are based on the detection of novel Edman degradation products generated from peptides in which phosphorylserine or phosphorylthreonine groups have been specifically modified; an example of …