Not only calmodulin (CaM) with Ca²⁺ regulates the activity of many enzymes and proteins, but also free-CaM (no Ca²⁺ bound) and Ca²⁺-independent CaM-binding proteins play roles in plant and animal cells. There is no in vivo method to identify the interaction between free-CaM and Ca²⁺-independent CaM-binding protein (CaMBP). Using site-directed mutagenesis by polymerase chain reaction (PCR), 5 mutant Arabidopsis calmodulin isoform 2 (AtCaM2) genes, mCaM2₁, mCaM2₁₂, mCaM2₁₂₃, mCaM2₁₂₄ and mCaM2₁₂₃₄ were obtained. The mutant mCaM2 encoded glutamine in place of glutamate (E32Q; E68Q; E105Q; E141Q) in one or more EF-hand Ca²⁺-binding motifs of AtCaM2. The recombinant mCaM2 proteins were produced in Escherichia coli, and subsequently separated on SDS-PAGE in the presence of Ca²⁺ or EGTA, their electrophoresis mobilities were related with that of mutant EF-hand motifs. ⁴⁵Ca²⁺ overlay analysis indicated that the more glutamate replaced by glutamine, the lower affinity with Ca²⁺ in the mCaM2 proteins. The mCaM2₁₂₃₄ mutant protein (E32Q; E68Q; E105Q;E141Q) was unable to bind Ca²⁺. Using yeast two-hybrid technique with mCaM2₁₂₃₄ as bait, it was possible to see interaction in Arabidopsis of AtCaM2 with IQD26, a calcium-independent CaM-binding protein. Site-directed mutation of AtCaM2 will aid the research of Ca²⁺, CaM and Ca²⁺-independent CaMBPs in plant biological processes.