The localization of fructose-1,6-bisphosphatase (FBPase) in human skeletal muscle was determined immunohistochemically using polyclonal antibodies. Light microscopy analysis, confirmed with the use of confocal microscopy, indicated that the enzyme is localized on both sides of the Z line of myocytes. The immunohistochemical investigation was confirmed by a co-sedimentation experiment which revealed that muscle FBPase binds strongly to alpha-actinin--a major structural protein of the Z line. This is the first report on localization of FBPase in skeletal muscle tissue.