Leuconostoc mesenteroides NRRL B-1355 grows on sucrose to produce two extracellular alpha-D-glucans. Although both are termed dextrans, they are chemically and physically distinct, and can be separated by fractional ethanol precipitation into fractions designated L and S. Fraction L is similar to B-512F dextran, having 95% alpha-(1 goes to 6) linkages and 5% alpha-(1 goes to 3) branch linkages, but fraction S has an alternating sequence of alpha-(1 goes to 6) and alpha-(1 goes to 3) linkages. Because of its structural differences from dextran, its different physical characteristics, and its resistance to hydrolysis by endodextranase, we have named glucan S, alternan, and the enzyme that synthesizes it from sucrose, alternansucrase. Alternansucrase has been isolated by two different methods. The first involves removal of the fraction L glucan from the culture fluid via hydrolysis by an endodextranase, followed by chromatography on Bio-Gel A5m. The void-volume fraction synthesizes only alternan, whereas the slower-migrating, second fraction synthesizes mainly dextran, together with some alternan. The second method utilized hydrophobic chromatography on O-(phenoxyacetyl) cellulose; a portion of the alternansucrase did not bind, whereas the bound portion, removed by eluting with detergent, contained both alternansucrase and dextransucrase. The glucans were identified by physical appearance, the concentration of ethanol required for precipitation, periodate-oxidation behavior, and susceptibility to hydrolysis by endodextranase. Also studied was the inhibition of the enzymes by 3-deoxy-3-fluoro-alpha-D-glucopyranosyl fluoride, tris(hydroxymethyl)aminomethane, 2-aminoethanol, and octyl beta-D-glucopyranoside.