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Escherichia coli grown at pH 5.0 became acid-tolerant (acid-habituated) but, in addition, neutralized medium filtrates from cultures of E. coli grown to log-phase or stationary-phase at pH 5.0 (pH 5.0 filtrates) induced acid tolerance when added to log-phase E. coli growing at pH 7.0. In contrast, filtrates from pH 7.0-grown cultures were ineffective. The pH 5.0 filtrates were inactivated by heating in a boiling water-bath but there was less activity loss at 75 °C. Protease also inactivated such filtrates, which suggested that a heat-resistant protein (or proteins) in the filtrates was essential for the induction of acid tolerance. Filtrates from cells grown at pH 5.0 plus phosphate or adenosine 3′:5′-cyclic monophosphate (cAMP) were much less effective in inducing acid tolerance, while the conversion of pH 7.0-grown log-phase cells to acid tolerance by pH 5.0 filtrates was inhibited by cAMP and bicarbonate. It seems likely that the acid tolerance response(acid habituation) involved the functioning of the extracellular protein(s) as protease reduces tolerance induction if added during acid habituation. Most inducible responses are believed to involve the functioning of only intracellular reactions and components; the present results suggest that this is not the case for acid habituation, as an extracellular protein (or proteins) is needed for induction.