Exponentially growing cells of Lactococcus lactis MG1363 are able to develop an Acid Tolerance Response (ATR) when incubated at pH 5, in both rich (M17)--and chemically defined (SA)--culture media. Physiological and proteomic characterization of this adaptive response indicated that L. lactis reorganizes its metabolism in response to acid stress to a great extent and quite differently in the two media. The development of ATR was fully dependent on protein de novo synthesis in SA and only partly dependent in M17. 2D gel electrophoresis revealed a total of 90 spots induced by acidity, 80 of which were identified by mass spectrometry. Only 10 proteins (BglA, PycA, GlmS, HasC, ArgS, GatA, AtpA, ArcB, Cfa, and SodA) were overproduced in the two media. A transcriptional analysis of the corresponding genes suggested that for half of them the mode of regulation may differ in the two media. Among the protein spots upregulated during the ATR in SA but not in M17, 13 already displayed an elevated rate of synthesis in M17 at neutral pH. These proteins could play an important role in the development of the protein de novo synthesis-independent ATR observed in M17.