The effect of liposomal encapsulation of ampicillin on the antibacterial activity against intracellular Listeria monocytogenes was studied by comparing survival of L. monocytogenes within peritoneal mouse macrophages in the presence of free ampicillin alone or in combination with liposome-entrapped ampicillin. In the presence of 50 micrograms of free ampicillin per ml of the incubation medium, intracellular growth of L. monocytogenes was still observed, although less as compared with intracellular growth in the absence of ampicillin. At a concentration of 50 micrograms of free ampicillin plus 100 micrograms of liposome-entrapped ampicillin per ml, 99% of the intracellular bacteria were killed. On the other hand, a concentration of 150 micrograms of free ampicillin per ml plus empty liposomes only inhibited intracellular bacterial growth, and the bacteria were not killed. In addition, empty liposomes at a concentration of 1 mumol of lipid per ml had no effect on intracellular bacterial growth. In broth, liposome-entrapped ampicillin at a concentration of 100 micrograms/ml was not bactericidal for L. monocytogenes, indicating that significant leakage of ampicillin from the liposomes with subsequent killing of the bacteria by the free drug did not occur. Therefore, we concluded that liposomal encapsulation of ampicillin results in an increased availability of the antibiotic for the intracellular bacteria.