Oligomerization of turkey gizzard myosin light chain kinase (MLCKase) was demonstrated by a zero-length cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC), a standard reagent used in investigations of specific protein-protein interaction [Mornet et al. (1989) J. Muscle Res. Cell Motil. 10, 10-24]. This approach revealed that in solution the kinase was not monomeric but the monomers were in equilibrium with the kinase dimeric and oligomeric forms. Addition of Ca2+/calmodulin (CM) shifted this equilibrium in the direction of the kinase dimers, accompanied by a 2-fold decrease of the kinase catalytic activity, in addition to a 2-fold decrease of its apparent affinity for CM [Sobieszek et al. (1993) Biochem. J. 295, 405-411]. The dimer (and/or oligomer) formation was shown to result from an interaction of the kinase autoinhibitory domain with its 24 kDa tryptic fragment containing titin-like domain II-3. The possible significance of the oligomerization in regulation of MLCKase activity is discussed.