Translocation of folded proteins by the Tat system of Escherichia coli is believed to rely on the presence of phosphatidylethanolamine (PE) and the negatively charged phospholipids cardiolipin (CL) and phosphatidylglycerol (PG). Here we show that while PE is indeed essential for activity, the Tat system is fully functional in a clsA/clsB/clsC deletion strain lacking CL, and in a pgsA deletion strain lacking both PG and CL during aerobic growth on complex media. In contrast to early studies that relied on strains with reduced lipid levels, this study therefore demonstrates that PG and CL are dispensable for Tat transport. The lack of these lipids may be compensated by other anionic phospholipids such as phosphatidic acid, CDP-diacylglycerol, or N-acyl-PE.