An investigation was made into the optimum conditions for the extraction and measurement of intestinal smooth muscle contractile proteins of laboratory rats. Isolation of the seromuscular layer was achieved by "mucosal stripping" (using a glass slide); isolation of contractile proteins were achieved by differential solubility (using high and low ionic buffers and ultracentrifugation). Histological evidence revealed that after mucosal striping all cells of the longitudinal and circular muscle layers of the seromuscular region remained intact. Assay of brush-border marker enzyme activities confirmed that mucosal striping did indeed remove the mucosa and there was very little villus contamination of the smooth muscle tissue preparations. Optimum physical conditions for the isolation of seromuscular and mucosal myofibrillary proteins were identified. We ascertained that considerable amounts of myofibrillarly proteins reside in the mucosa, but could be adequately separated by mucosal striping. Improved recoveries of purified contractile proteins necessitated the inclusion of protease inhibitors during all processing steps. Using the optimum method, polyacrylamide gel electrophoresis of contractile proteins showed that the predominant smooth muscle contractile proteins, i.e., myosin heavy chain, actin, tropomyosin, and myosin light chains, were indeed preferentially isolated by our methodology. Using these techniques we demonstrated that the synthesis rates of intestinal contractile proteins were reduced by acute ethanol dosage. These results may be responsible for, or reflect, alcohol-induced defects in intestinal motility. & 1993 Academic Press, Inc.