We have studied the immunological and structural properties of myosin light-chain kinases. Immunological studies were performed with affinity-purified antibodies to turkey gizzard smooth-muscle myosin light-chain kinase. Immunoprecipitation experiments demonstrated that avian smooth muscles contain a myosin light-chain kinase of Mr 130,000, whereas the enzyme immunoprecipitated from canine smooth muscles tested has an Mr of 150,000. These antibodies do not react with cardiac- or skeletal-muscle myosin light-chain kinases. Experiments performed with myosin light-chain kinases purified from turkey gizzards (Mr 130,000), bovine tracheal smooth muscle (Mr 160,000) and human platelets (Mr 100,000) demonstrated the following: the primary structures of the turkey gizzard and bovine tracheal enzymes appear to be quite different, based on one-dimensional peptide maps; only one-third as many antibodies bind to the bovine tracheal enzyme as compared to the turkey gizzard enzyme; the antibody:myosin light-chain kinase ratios for half-maximal inhibition of all three enzymes are similar. Based on these data, we conclude that myosin light-chain kinases constitute an immunologically and structurally heterogeneous group of enzymes that have certain catalytic and regulatory properties in common.