Primary cultures prepared from embryonic chick pectoral muscle were subjected to heat shock, and the effect on acetylcholinesterase activity in the cultures was examined. A rapid recovery in enzyme activity was observed soon after an initial heat shock-induced drop and was shown to be independent of de novo synthesis of protein, since it could occur in the presence of an inhibitor of protein synthesis. Lectin binding and sucrose gradient centrifugation studies suggested that molecular monomers and dimers found in the endoplasmic reticulum are involved in the observed recovery of acetylcholinesterase activity. Enhanced activation of a preexisting pool of inactive enzyme was clearly not the main agent of the recovery in enzymic activity. Recovery relied principally on restoration of the activity of previously active, heat-denatured acetylcholinesterase molecules found in the endoplasmic reticulum. Possible agents involved in the recovery of enzymatic activity might be heat shock proteins acting as molecular chaperones.