Abstract Phospholipid-protein interactions have been investigated in a phospholipid-requiring enzyme, FAD-dependent malate dehydrogenase isolated from Mycobacterium smegmatis membranes, to correlate these interactions with enzyme function. The ability of several natural and synthetic phospholipids including CL and PE, which are major phospholipids in M. smegmatis membranes, to activate purified, lipid-depleted, enzymatically inactive malate dehydrogenase was examined. Anionic phospholipids and PE activated the enzyme, while zwitterionic phospholipids did not. A PE/PC mixture activated the enzyme in the form of both bilayer and non-bilayer structure. CL/PE mixtures activated malate dehydrogenase much more than each single phospholipid species. All anionic phospholipids used stabilized the enzyme, while PE and zwitterionic phospholipids did not. CL and a CL/PE mixture protected malate dehydrogenase from proteinase digestion, while PE did not. All phospholipids and phospholipid mixtures tested caused little secondary structural change in malate dehydrogenase. The results obtained in this study suggest that CL and CL/PE mixtures could form stable, enzymatically active complexes with malate dehydrogenase which might be similar to the native complex in M. smegmatis membranes. Although PE could activate malate dehydrogenase in both bilayer and non-bilayer form, it formed a complex with malate dehydrogenase which was inferior in terms of stability and susceptibility to proteinases, indicating that PE alone poorly reconstitutes the active enzyme-phospholipid complex.