The aim of this work was to use two approaches (microbiological and proteomic) to study the effect of cold adaptation (3 h at 4 degrees C) on the survival of two Escherichia coli strains, I2 and R3, following freezing at -20 degrees C for 24 h and thawing for 45 min at 37 degrees C. The effect of cold adaptation on cell survival after freezing was determined by measuring viable counts on selective (PTX) and nonselective media (TSA). The beneficial effect of the cold treatment was more pronounced for the freezing-sensitive R3 strain: Prior to adaptation, differences between the two media were 3.5 log10 for R3 and 0.4 log10 for I2. After adaptation, the differences were 2.5 log10 and 0.1 log10 for R3 and I2, respectively. The proteins from two cell compartments, cytoplasm and outer membrane, were separated by two-dimensional electrophoresis and identified by mass spectrometry or Edman sequencing. The adaptation resulted in changes in the expression of certain proteins. Among the principal changes to protein profiles in strain R3 following cold adaptation, we observed an over-expression of the EF-TU elongation factor in the outer membrane, and an under-expression of flagellin (FLIC) in the cytoplasm. Very few changes were observed in strain I2.