The response of Mycobacterium smegmatis to a cold shock was investigated by monitoring changes in both growth and cellular protein composition of the organism. The nature of the cellular response was influenced by the magnitude of the temperature reduction, with the shock from 37°C to 10°C having the most widespread effect on growth, metabolism and protein composition. This 27°C temperature reduction was associated with a lag period of 21–24 h before increases were seen in all the measured cellular activities. The response to cold shock was adaptive, with growth resuming after this period, albeit at a 50-fold slower rate. The synthesis of at least 15 proteins was induced during the lag period. Two distinct patterns of cold-induced synthesis were apparent, namely transient and continuous, indicating the production of both cold-induced and cold-acclimation proteins. One of these cold-shock proteins, CipMa, was identified as the histone-like protein, Hlp, of M. smegmatis, which is also induced during anaerobic-induced dormancy. The corresponding gene demonstrated transient, cold-inducible expression with a five- to sevenfold increase in mRNA occurring 9–12 h after temperature shift. Although bacterial survival was unaffected, CipMa/Hlp knock-out mutants were unable to adapt metabolically to the cold shock and resume growth, thus indicating a key role for CipMa in the cold-shock response.