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A mutant strain of Saccharomyces cerevisiae (D10-ER1) has been isolated after a two-step mutagenesis of strain 4059-358D (SUC 1) using ethyl methane sulfonate. Cells of this new strain produced a level of total invertase equaling that of 4059 but contained only trace amounts of the small, internal, aglycan form of the enzyme (less than 0.1% of total in D10-ER1 compared with 6% in 4059). When D10.ER1 was crossed with an invertase-hyperproducing strain dgr3 (SUC3), progeny were isolated (HZ400-5A and HZ400-2C) in which levels of total invertase had at least quadrupled. The percentage of small invertase, however, remained insignificant. Levels of small invertase in strain HZ400-5A were determined by affinity chromatography on conconavalin A-Sepharose, gel permeation chromatography, and isopycnic centrifugation in CsCl. The large invertase of the SUC1 yeasts described here was found to contain a form apparently greater in size than the large invertase of the SUC2 strain FH4C; this probably reflects a higher content of carbohydrate. The overall results of this study do not support a direct structural relationship between large and small invertases. The implications on invertase biosynthesis and structure are discussed.