Current non-viral DNA vectors for gene therapy are limited by low cellular transfection efficiencies and low levels of gene expression due to inefficient endosomal DNA release. We have used perfringolysin O (PFO), a membrane active bacterial protein, to deliver DNA into cells. PFO belongs to the so-called sulphydryl-activated family of membrane active bacterial proteins, which have been used to deliver small molecules and proteins into cells. PFO was incorporated into DNA complexes through a biotin-streptavidin bridge and the DNA-PFO complexes were used to deliver the Escherichia coli beta-galactosidase gene into cells. High levels of gene expression were achieved in murine sol 8 myoblast cells using these DNA-PFO complexes. The level of gene expression correlated well with the content of PFO in the complexes. Under optimal conditions, 15-20% of the cells were stained blue with X-gal. Furthermore, the expression was independent of a receptor ligand. Thus, membrane active bacterial proteins may be an important tool for the future development of non-viral DNA delivery systems for gene therapy.