Here we report the characterization and optimization of a peptide/non-cationic lipid gene delivery system that successfully produces high levels of gene expression when delivered by microinjection into chicken embryos in vivo. In addition to plasmid DNA, the delivery complex consisted of four components: 1) a "condensing" peptide with both hydrophobic and cationic amino acid segments; 2) a "fusogenic" peptide with both membrane insertion and amphipathic helical segments; 3) a relatively short-chain phosphatidylcholine (14:1 cis-9); and 4) polyethyleneglycol conjugated to dioleoylphosphatidylethanolamine through a disulfide linkage. Optimum amounts of each component were determined by measuring expression of a luciferase reporter gene following a 24-hour incubation with chick embryo fibroblast (CEF) cells in culture. When relatively low amounts of condensing peptide, fusogenic peptide, or lipid were assembled into the complexes, relatively large concentrations of complex were required to reach maximum gene expression. When the amounts of peptide or lipid were increased, less complex was required to achieve maximum expression, but expression fell substantially with higher amounts of added complex. The polyethyleneglycol component significantly increased gene expression. With some preparations, luciferase activities in the CEF cells reached 1x10(10) relative light units per second per mg protein within 24 hours. Following the optimization experiments with the CEF cells, formulations containing low levels, intermediate levels, and high levels of the delivery system components were assembled with green fluorescent protein plasmid DNA, then microinjected into somite regions of chicken embryos in vivo. It was found that intermediate levels of the components gave the most reliable formulations for inducing localized gene expression in the somitic cells.