CaM[3 TnC] is a calmodulin-cardiac troponin C chimeric protein containing the first, second, and fourth calcium-binding domains of calmodulin (CaM) and the third calcium-binding domain of cardiac troponin C (cTnC) (George, S. E., Su, Z., Fan, D., and Means, A. R. (1993) J. Biol. Chem. 268, 25213-25220). CaM[3 TnC] shows altered activation of phosphodiesterase (PDE) and is a potent competitive inhibitor of smooth muscle myosin light chain kinase (smMLCK) activation by CaM. To determine why CaM[3 TnC] exhibits altered target enzyme interactions, we constructed a series of domain 3 CaM mutants. We began with subdomain substitutions, replacing most of CaM's helix 5, Ca2+ binding loop 3, and helix 6 with the corresponding subdomains of cTnC. Only CaM[helix 6-TnC] exhibited significant impairment of smMLCK and PDE activation. We then individually substituted the residues in the region of CaM's helix 6 with the corresponding cTnC residue. This revealed that CaM residues Thr-110, Leu-112, and Lys-115 were critical for full smMLCK activation and could not be substituted by the corresponding cTnC residue (Gln, Thr, and Thr, respectively). In contrast, only the L112T substitution significantly affected PDE activation. The CaM-smMLCK peptide structure (Meador, W. E., Means, A. R., and Quiocho, F. A. (1992) Science 257, 1251-1255) suggests a relationship between the proposed helix 6 smMLCK-activating residues and those previously described in helix 2 (VanBerkum, M. F. A., and Means, A. R. (1991) J. Biol. Chem. 266, 21488-21495).