Nisin production by Lactococcus lactis CECT 539 was followed in batch cultures in whey supplemented with different concentrations of glucose and in two realkalized fed-batch fermentations in unsupplemented whey, which were fed, respectively, with concentrated solutions of lactose and glucose. In the batch fermentations, supplementation of whey with glucose inhibited both the growth and bacteriocin production. However, fed-batch cultures were characterized with high productions of biomass (1.34 and 1.51 g l(-1)) and nisin (50.6 and 60.3 BU ml(-1)) in comparison to the batch fermentations in unsupplemented whey (0.48 g l(-1) and 22.5 BU ml(-1)) and MRS broth (1.59 g l(-1) and 50.0 BU ml(-1)). In the two realkalized fed-batch fermentations, the increase in bacteriocin production parallels both the biomass production and pH drop generated in each realkalization and feeding cycle, suggesting that nisin was synthesized as a pH-dependent primary metabolite. A shift from homolactic to heterolactic fermentation was observed at the 108 h of incubation, and other metabolites (acetic acid and butane-2,3-diol) in addition to lactic acid accumulated in the medium. On the other hand, the feeding with glucose improved the efficiencies in glucose, nitrogen, and phosphorus consumption as compared to the batch cultures. The realkalized fed-batch fermentations showed to be an effective strategy to enhance nisin production in whey by using an appropriate feeding strategy to avoid the substrate inhibition.