Cyclin-dependent kinase 5 (Cdk5) has been shown to regulate adhesion and migration of lens and corneal epithelial cells. To explore protein-protein interactions that may mediate these functions, we performed yeast two-hybrid screening on an embryonic rat lens library using Cdk5 and its regulators, p35 and p39 as baits. This screen identified an interaction between p39 and non-muscle myosin essential light chain (MLC17). GST pull-down experiments demonstrated that p39 binds directly to MLC17 through a strong binding site in the N-terminal 109 amino acids of p39. Immunoprecipitation of proteins from Cost cells co-transfected with GFP-MLC17 and HA-p39 confirmed that these proteins interact intracellularly. Immunofluorescence microscopy of co-transfected lens epithelial cells showed that GFP-MLC17 and HA-p39 co-localize along cytoskeletal fibrils. Moreover, endogenous rat lens p39 co-immunoprecipitated with MLC17 and myosin heavy chain II (MHC II), demonstrating that the interaction is physiological and serves to link p39 to the cytoskeleton. Published by Elsevier Inc.