Background: Myosin light chain kinase (MLCK) specifically phosphorylates the myosin regulatory light chain in a calcium/calmodulin (Ca2+/CaM)‐dependent manner in animal cells. The roles of MLCK are not fully understood, particularly in nonmuscle cells. Therefore, we cloned and characterized a Drosophila MLCK gene as the first step in a genetic analysis of this process. Results: Four transcripts are produced from this gene. These transcripts encode at least three isoforms (isoform‐I, ‐II and ‐III), which share a kinase domain, a fibronectin type III motif and an immunoglobulin C2 motif. However, regulatory regions differ between isoform‐I/II and ‐III due to the alternative splicing of the exon encoding a CaM‐binding domain. As a result, isoform‐I and ‐II are Ca2+/CaM‐dependent forms, whereas isoform‐III is a Ca2+/CaM‐independent form. Northern blotting and in situ hybridization showed that the expressions of these isoforms are distinctly regulated in stage‐ and tissue‐dependent manners. Isoform‐I seems to be expressed ubiquitously, while isoform‐III is expressed predominantly in muscle tissues. In contrast to these isoforms, isoform‐II is specific to late pupa and adult. Conclusion: In Drosophila, a single MLCK gene produces multiple isoforms whose regulatory regions and expression patterns are different. These differences suggest various cellular functions of MLCK in Drosophila.