The interaction of ANS with Escherichia coli cells, isolated cell walls, total cell envelopes and inner membranes was investigated in the presence and absence of Ca2+ ions. The addition of Ca2+ to intact cells at room temperature did not result in enhancement of fluorescence intensity. On the contrary the addition of Ca2+ to intact cells at 2 degrees C resulted in a progressive increase of fluorescence intensity with the maximum reached approximately by the 20th minute. The addition of Ca2+ to different membrane preparations showed a two-fold increase. Addition of Ca2+ to spheroplast membrane preparations did not result in any increase of the number of binding sites for ANS. There was a four-fold increase in quantum yields. All membrane preparations in titrations with Ca2+ showed saturation kinetics. The obtained results are discussed in terms of structural alterations in E. coli membranes and in relation with biological effects.