1. The effects of Mg2+ on the fluidity and on the transport properties of mitochondrial inner membrane were compared in parallel experiments. The fluidity was measured by intercalated fatty acid spin probes. Valinomycin-induced K+ uptake was followed using an ion-selective electrode. 2. The rotational diffusion rate of lipids was very slightly affected by Mg2+, whereas the ordering of the probed region of the inner membrane increased considerably above 30 degrees C in the presence of Mg2+. Mg2+ strongly inhibited K+ transport, particularly above 30 degrees C. 3. In the presence of different concentration of MgCl2 (0--30 mM) the order parameter showed no significant variation, whereas the rotational correlation time had essentially biphasic character with a minimum (i.e., faster diffusion rate) at 10 mM MgCl2. 4. We conclude that Mg2+ induces structural changes in the mitochondrial inner membrane and concomitant changes in its functional properties. The term 'fluidity' is inadequate for the interpretation of the data, since changes in the order parameter and in the characteristic correlation time of the inner membrane upon addition of Mg2+ did not show parallel tendencies.