Neurogranin (NG) binding of calmodulin (CaM) at its IQ domain is sensitive to Ca2+ concentration and to modifications by protein kinase C (PKC) and oxidants. The PKC phosphorylation site of NG is within the IQ domain whereas the four oxidant-sensitive Cys residues are outside this region. These Cys residues were oxidized forming two pairs of intramolecular disulfides, and could also be glutathiolated by S-nitrosoglutathione resulting in the incorporation of four glutathiones per NG. Circular dichroism (CD) showed that modification of NG by phosphorylation, oxidation forming intramolecular disulfides, or glutathiolation did not affect the R-helical content of this protein. Mutation of the four Cys residues [Cys (-)-NG] to Gly and Ser did not affect the R-helical content either. Interaction of CaM with the reduced (red), glutathiolated (GS)-, or Cys(-)-NG in the Ca2+-free solution resulted in an increase in the R-helicity determined by their CD spectra, but relatively little change was seen with the oxidized NG (ox-NG) or phosphorylated NG (PO4-NG). The binding affinities between the various modified forms of NG and CaM were determined by CD spectrometry and sedimentation equilibrium: their affinities were Cys (-)-NG > red-NG, GS-NG > ox-NG > PO4-NG. Unlike Cys (-)-, red-, and GS-NG, neither ox-nor PO4-NG bound to a CaM-affinity column. Thus, both oxidation of NG to form intramolecular disulfides and phosphorylation of NG by PKC are effective in modulating the intracellular level of CaM. These results indicate that modification of NG to form intramolecular disulfides outside the IQ domain provides an alternative mechanism for regulation of its binding affinity to CaM.