Interaction of mitochondrial creatine kinase (mtCK) with either synthetic or natural zwitterionic or acidic phospholipids was monitored by surface pressure measurements. Injection of mtCK beneath a monolayer at very low surface pressure results in a large increase in the apparent area per lipid molecule reflecting the intrinsic surface activity of the protein. This effect is particularly pronounced with anionic phospholipid-containing films. Upon compression to high lateral pressure, the protein is squeezed out of the lipid monolayer. On the contrary, mtCK injected beneath a monolayer compressed at 30 mN/m, does not insert into the monolayer but is concentrated below the surface by anionic phospholipids as evidenced by the immediate and strong increase in the apparent molecular area occurring upon decompression. Below 8 mN/m the protein adsorbs to the interface and remains intercalated until the lateral pressure increases again. The critical pressure of insertion is higher for anionic lipid-containing monolayers than for films containing only zwitterionic phospholipids. In the former case it is markedly diminished by NaCl. The adsorption of mtCK depends on the percentage of negative charges carried by the monolayer and is reduced by increasing NaCl concentrations. However, the residual interaction existing in the absence of a global negative charge on the membrane may indicate that this interaction also involves a hydrophobic component.