Three skeletal muscles viz., gastrocnemius, pectoralis and diaphragm from rats acclimated to a low temperature (4 +/- 1 degrees C; 16 hr daily; maximum for 8 weeks) exhibit an increased myosin ATPase activity. An analysis of native myosin from these muscles under non-dissociating conditions reveals two myosin isozymes instead of a single isozyme expressed in control muscles. Isoelectric focusing (IEF) coupled with two dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE) confirms an increased phosphorylation of myosin light chain 2 (MLC2) in muscles from cold acclimated rats.