A domain in the inducible, macrophage nitric oxide (NO) synthase has been selected as the putative calmodulin-binding site. The domain was synthesized as a peptide of 29 residues [P29, NO synthase-(504-532)-peptide], having the accepted hydrophobic/basic composition of calmodulin-binding domains and containing, like most of them, an aromatic amino acid at its N-terminus and a long chain aliphatic residue 12 amino acids downstream of it. A 34-residue peptide from the synthase sequence [P34, NO synthase-(499-532)-peptide], consisting of peptide P29 and of the five extra N-terminal amino acids, three of them basic, was also synthesized. Both peptides bound calmodulin in the presence as well as in the absence of Ca2+ (i.e. in the presence of excess EGTA). The KD of the binding in the presence of Ca2+ was < or = 1 nM. The binding affinity was lower, but still remarkably high in the presence of EGTA. The peptides counteracted the stimulation by calmodulin of a classical calmodulin-target enzyme, the Ca2+ pump of the plasma membrane.