Human serum inhibits the antibacterial activity of colistin and polymyxin B against Pseudomonas aeruginosa in vitro. By use of a colony-count assay, the inhibitory factor in serum was identified as calcium. The bactericidal activity of colistin was inhibited by either 10−3m CaCl2, 15% human serum, or 15% heparinized plasma. Colistin was not inhibited by plasma containing chelating agents. Colistin may be less active in vivo than predicted on the basis of in-vitro tests of susceptibility to the antibiotic.