Antisense oligodeoxynucleotides (ODNs) possess great potential as sequence-specific therapeutic agents. Sufficient concentrations of intact ODN must bypass membrane barriers and access the cytosol and nucleus, for ODNs to be therapeutically effective. A cytosolic delivery strategy was designed to improve the efficiency of ODN delivery in bone-marrow-derived macrophages. This liposome-based formulation utilizes listeriolysin O (LLO), the endosomolytic hemolysin from Listeria monocytogenes, to mediate the escape of ODN from endocytic compartments into the cytosol. To monitor the cytosolic delivery of ODN, subcellular trafficking of fluorescently labeled ODNs was visualized using epifluorescence microscopy. The expression of target protein and mRNA after delivery was measured using flow cytometry and Northern blot analysis, respectively. ODN specific for murine intercellular adhesion molecule-1 (ICAM-1) encapsulated in LLO-liposomes was released to the cytosol and trafficked to the nucleus, efficiently and specifically suppressing activation-induced expression of ICAM-1 at both protein and mRNA levels. Delivery without LLO resulted in sequestration of ODN in vesicular compartments leading to little inhibition of ICAM-1 expression, which supports the requirement of LLO for efficient cytosolic delivery using this system. The data clearly demonstrate that LLO-mediated escape of ODN from intracellular vesicles is an effective approach to achieve full therapeutic antisense activity in cultured macrophages.