Thirteen aminoglycoside antibiotics were investigated with respect to their ability to replace Ca from phosphatidylserine monolayers. The affinities of the glycosides for the Ca-binding sites depended on the pH and the Ca2+ concentration of the aqueous subphase. At a Ca2+ concentration of 1.2 X 10(-5)M and a pH of 7.5 the half maximum concentrations to replace Ca were found to range from 0.4 X 10(-6) (sisomicin) to 6 X 10(-6)M (streptomycin). The interaction between the streptomycins and Ca were of a competitive nature, whereas the aminoglycoside antibiotics with higher affinities displayed unusual steep dose-response curves suggesting positive cooperativity. The aminoglycoside antibiotics were also able to replace Ca from biomembranes (red cell ghosts and isolated sarcolemma of cardiac muscle) in a dose-dependent manner. The high affinity of aminoglycoside antibiotics to a phospholipid is considered to be involved in the toxic side effects of these antibiotics, e.g. nephrotoxicity and impairment of contractile force.