The factors of bile tolerance (as one among the fundamental characteristics of probiotic bacteria) were determined in lactococci by using plasmid variants. Bile tolerance of Lactococcus lactis wild-type (WT) strains 527 and N7 (determined by viability counts on bile-containing agar) was equivalent to the corresponding plasmid-free derivatives. In contrast, L. lactis WT strain DRC1 had lower bile tolerance than its plasmid-free derivative DRC1021. Plasmid pDR1-1B, extracted from strain DRC1, was introduced into strain DRC1021 by co-transformation with the vector plasmid pGKV21 as an indicator. Strain DRC121 (DRC1021 harboring pGKV21) had good bile tolerance as did strain DRC1021, while strain DRC13 (DRC1021 harboring both pDR1-1B and pGKV21) did not. Fatty acid (FA) composition was different between strains DRC121 and DRC13. The plasmid pDR1-1B or plasmid profile and FA composition are key factors for bile tolerance of strain DRC1, and therefore changing the plasmid profile might be a way of modulating bile tolerance in lactococci.