The results here are the first clear demonstration of a physiological role for cytosolic Ca(2+) in Escherichia coli by releasing a Ca(2+) binding protein, apoaequorin, from inclusion bodies. In growth medium LB the cytosolic free Ca(2+) was 0.1-0.3 microM. Addition of EGTA reduced this to <0.1 microM, whereas addition of Ca(2+) (10mM) resulted in a cytosolic free Ca(2+) of 1-2 microM for at least 2h. Ca(2+) caused a 1.5- to 2-fold increase in the level of apoaequorin induced by IPTG. Whereas EGTA induced a 50% decrease. The effect of a Ca(2+) was explained by release of protein from the inclusion bodies, together with a stabilisation of apoaequorin against degradation. Ca(2+) also reduced the generation time by 4-5 min. These results have important implications for unravelling the physiological role of cytosolic Ca(2+) in bacteria, particularly where several species are competing for the same nutrients, such as in the gut.