Filaggrin and a phosphorylated form of filaggrin, which has been shown by pulse-chase studies to be a precursor form of the protein [Dale, B. A., & Ling, S. Y. (1979) Biochemistry 18, 3539-3546], were compared for functional, biochemical, and physical properties. Filaggrin reacts with keratin filaments to form visible macrofibrils, unlike the precursor which does not. Biochemical and peptide-mapping studies suggest that the two proteins have similar, perhaps identical, amino acid sequences. The major differences between the two proteins are in molecular weight (precursor, 44 200 g/mol; filaggrin, 38 400 g/mol), the existence of oligomeric forms of the precursor, and the presence of phosphate in the precursor (15-20 mol/mol of protein). Phosphoserine was identified in the precursor, but neither phosphothreonine nor phosphotyrosine was observed. The results of proteolytic digests of [32P]phosphate-radiolabeled precursor show that the phosphate is unevenly distributed throughout the molecule and may be localized in approximately 30% of the precursor. A discrete localization of the phosphate in the precursor may block a specific keratin filament combining site and so prevent premature aggregation of these filaments during epidermal differentiation. It is suggested that a specific phosphatase is involved in the dephosphorylation, because several phosphatases of general specificity, including rat epidermal lysosomal acid phosphatase, did not catalyze this conversion.