In this paper, we describe three approaches to study the single trimethyllysine 115 in calmodulin. First, 14N NMR spectroscopy has been used as a novel spectroscopic tool. Because of the unique symmetrical tetrahedral substitution of its side chain, the trimethyllysine residue gives rise to a sharp 14N NMR resonance; hence, this has allowed the detection and quantitation of the level of trimethylation. Trimethyllysine side chains of bovine testis calmodulin and yeast cytochrome c were shown to have a high mobility in aqueous solution as determined by 14N NMR relaxation measurements. Second, we have purified mammalian calmodulin from an overproducing Escherichia coli strain. By comparison of the 1H-13C heteronuclear multiple quantum coherence spectra of 13C-dimethylated calmodulin samples from bovine testis and E. coli, the resonance for Lys-115 in bacterially expressed calmodulin could be identified. pH titration experiments showed that epsilon-NH2 group of Lys-115 has a normal pKa value both in the apo and Ca2+ forms of the protein and in a complex of calmodulin with a 22-residue calmodulin-binding peptide derived from myosin light chain kinase. Third, we have shown that mutation of Lys-115 to the uncharged Gln residue does not alter the ability of the protein to stimulate the enzymes cyclic nucleotide phosphodiesterase and myosin light chain kinase. These results show that the trimethylation of Lys-115 is not caused by an unusual pKa and reactivity of its epsilon-NH2 group and that its side chain remains flexible. Moreover, our data suggest that the introduction of a permanent positive charge on Lys-115 by trimethylation is also not the major reason for this specific post-translational modification.