The exchange of 17-kDa essential light chain (LC17) in smooth muscle myosin was carried out by incubating myosin with a 10-fold molar excess of exogenously added LC17 over the corresponding endogenous light chain in the presence of trifluoperazine and 4.5 m ammonium chloride. Porcine aorta myosin contains two kinds of LC17 isoform, LC17nm and LC17gi, while chicken gizzard myosin contains only one kind of LC17 isoform. As LC17gi can be separated from LC17nm and gizzard LC17 by urea-gel electrophoresis, LC17nm in aorta myosin and LC17 in gizzard myosin were exchanged with LC17gi and LC17gi in aorta myosin was exchanged with LC17nm, and the degree of exchange was estimated by urea-gel electrophoresis. Under the optimal conditions (6 and 10 degrees C for aorta and gizzard myosin, respectively), nearly 90% of exchange, which is close to the theoretical value, was achieved for the former combinations, and a slightly lower exchange was obtained for the latter. The LC17-exchanged myosins contained stoichiometric amounts of the heavy and light chains and retained the original nature in the phosphorylation-dependent actin-activated ATPase activity, 6S-10S conformational transition, and filament assembly.