A genetic library of Streptococcus mutans GS-5, constructed in an Escherichia coli plasmid vector, was screened for cells which could utilize sucrose as the sole carbon and energy source. The recombinant plasmid pFRU1, containing a 4.2-kilobase pair insert of S. mutans DNA, was shown to confer this phenotype. Further characterization of the gene product encoded by pFRU1 revealed that the enzyme was a beta-D-fructosidase with the highest specificity for the beta (2----6)-linked fructan polymer levan. The enzyme could also hydrolyze inulin [beta (2----1)-linked fructan], sucrose, and raffinose with 34, 21, and 12%, respectively, of the activity observed for levan. The gene (designated fruA) appeared to be expressed under its own control in E. coli, as judged by the lack of influence on gene product activity of induction or repression of the beta-galactosidase promoter adjacent to the insertion site on the cloning vector. The protein was purified to homogeneity, as judged by silver staining of purified protein in denaturing and reducing conditions in polyacrylamide gels, from sonic lysate of E. coli, as well as from culture supernatants of S. mutans GS-5 grown in a chemostat at low dilution rate with fructose as the sole carbohydrate source. Both purified proteins had an apparent molecular mass of 140,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were immunologically related and comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as determined by Western blotting with antisera raised against the cloned gene product, and were identical in all physical and biochemical properties tested. The pH optimum of the enzyme acting on fructan polymers was 5.5, with a significant amount of activity remaining at pH 4.0. The optimum pH for sucrose degradation was broader and lower, with a peak at approximately 4.5. Enzyme activity was inhibited almost completely by Hg2+ and Ag2+, inhibited partially by Cu2+, not inhibited by fluoride ion or Tris, and slightly stimulated by Mn2+ and Co2+. Fructan polymers were attacked exohydrolytically by the enzyme, fructose being the only product released. With sufficient time, both levan and inulin were degraded to completion, with no evidence of product inhibition.