Alternan-producing Leuconostoc mesenteroides strain NRRL B-1355 and its glucansucrase-negative derivative NRRL B-21414 were transformed by electroporation using four Gram positive-Gram negative shuttle vectors. Optimal conditions were 400 Ω and 10 kV cm−1, resulting in transformation efficiencies of up to 3.5×104 per μg DNA. Relatively low copy numbers and native plasmids made it difficult to visualize the introduced plasmids on ethidium bromide-stained gels and, in some cases, on blot hybridizations. However, PCR analysis indicated that 95% of putative transformants carried plasmid sequences. Direct colony PCR was shown to work well for this system and also for transformants of L. mesenteroides subsp. cremoris.