Alkaline phosphatase is normally localized to the periplasm of Escherichia coli and is unable to fold into its native conformation if retained in the cytoplasm of growing cells. The alkaline phosphatase activity of E. coli expressing a version of the protein without a signal sequence was nonetheless found to increase gradually when the growth of cells was suspended. At least 30% of the protein was activated over the course of several hours when freshly grown exponential-phase cells were held on ice. Similar behavior was observed with cells expressing certain other mutant versions of alkaline phosphatase that are retained in the cytoplasm. The activation resulted not from the passage of the alkaline phosphatase into the periplasm but from the slow folding of alkaline phosphatase into its native conformation in the cytoplasm. These findings indicate that the mechanism by which proteins are normally kept reduced in the cytoplasm fails to function if cells are not growing. It was found that the addition of the sulfhydryl-alkylating agent iodoacetamide to cells after growth blocks this activation completely. This treatment can therefore diminish the likelihood of spurious enzyme activity measurements in studies that make use of alkaline phosphatase fusion proteins.