Actinomyces viscosus ATCC 15987 was examined for its ability to hydrolyze its own levan. Washed whole cells and an ammonium sulfate fraction from cell-free culture fluids were shown to possess levan hydrolase activity. Analyses of reaction mixtures by gel filtration and thin-layer chromatography demonstrated that the product of levan hydrolysis was free fructose. The cell-associated and extracellular enzyme preparations also hydrolyzed inulin and the levans synthesized by Aerobacter levanicum and Bacillus subtilis. Growth of A. viscosus in media supplemented with 0.1% A. viscosus levan resulted in a 33-fold increase and a 7-fold increase in the specific activities of the respective extracellular and cell-associated enzymes when compared with those from 55 mM glucose cultures. Growth in the presence of 29.2 mM sucrose resulted in a 28-fold increase and a 5-fold increase in the specific activities of the respective enzymes when compared with those from the glucose cultures. The extracellular enzyme exhibited high activity over a wide pH range, with 87 and 89% of its pH 6.0 optimum activity at pH 5.0 and 7.0, respectively. The cell-associated enzyme also exhibited optimum activity at pH 6.0, but this was decreased to 10 and 20% at pH 5.0 and 7.0, respectively. Analysis for the presence of extracellular levan during growth of A. viscosus in sucrose broths demonstrated that peak levan concentrations occurred during the mid-exponential to late-exponential phase of growth followed by a rapid decline in extracellular levan as a result of levan hydrolase activity.