Myosin light chain kinase regulates the activity of myosin by phosphorylating the myosin regulatory light chain. Here we describe the cloning and characterization of cDNAs encoding Drosophila melanogaster myosin light chain kinase. We amplified a fragment of the Drosophila mlck gene using degenerate primers homologous to a highly conserved region in myosin light chain kinase proteins of vertebrate species. We used the gene fragment to isolate corresponding Drosophila mlck cDNAs. The deduced protein sequence of the cDNAs shows high homology to the catalytic and regulatory domains of vertebrate nonmuscle, smooth muscle and skeletal muscle myosin light chain kinase. Protein motifs I and II, which are present in vertebrate nonmuscle and smooth muscle myosin light chain kinase, but not in skeletal muscle myosin light chain kinase, are also present in Drosophila myosin light chain kinase. Transcript and cDNA analysis shows the gene encodes multiple messages and is expressed in nonmuscle and muscle cells, including the adult indirect flight muscle. Genomic Southern analysis and chromosome hybridization suggest mlck is a single copy gene which maps to chromosome band 52D, and is not haplo-insufficient for flight.