All of the superoxide dismutase isozymes of Escherichia coli have been shown to occur in the cell matrix, and none have been found in the periplasm. This was the case with both E. coli B and E. coli K-12, whether grown on a low phosphate medium or on a Trypticase soy-yeast extract medium. Alkaline phosphatase was used as a marker of the periplasm; adenosine deaminase and glucose 6-phosphate dehydrogenase were used as matrix markers, and consistent results were obtained by osmotic shock, spheroplast formation, and use of a diazonium salt that penetrates the periplasm but cannot cross the plasma membrane. A previous report that the iron-containing superoxide dismutase of E. coli is a periplasmic enzyme is now seen to have been in error.