E3 colicinogenic cells are immune to colicin E3. A protein called "E3 immunity protein" was previously isolated from E3 colicinogenic cells and shown to prevent the E3-induced in vitro inactivation of ribosomes. We now show that the structural gene for E3 immunity protein resides on the Col E3 plasmid, a plasmid which is present in E3 colicinogenic cells and carries the structural gene for colicin E3 production. For this purpose, Col E3 plasmid DNA was purified and characterized. The DNA preparation was shown to be homogeneous as judged by electron microscopy as well as agarose gel electrophoresis and has the ability to transform noncolicinogenic Escherichia coli cells into E3 colicinogenic cells with a high efficiency. The Col E3 DNA was then used as a template in a DNA-dependent in vitro protein-synthesizing system, and protein products were characterized. A radioactive protein product was detected which co-migrates with reference E3 immunity protein in urea-polyacrylamide gel electrophoresis at pH 8.7 and urea-sodium dodecyl sulfate polyacrylamide gel electrophoresis at pH 7.6. This protein was produced only in the presence of Col E3 plasmid DNA. No such protein was produced when Col E3 plasmid DNA was omitted or replaced with chromosomal DNA. This radioactive protein was isolated and shown to be very similar to reference E3 immunity protein, as judged by the correspondence of tryptic peptides. The synthesis of immunity protein in vitro was also shown by radioimmunodiffusion. Thus, the structural gene for E3 immunity protein resides on the Col E3 plasmid. In addition, we have shown that colicin E3 protein is also synthesized in the same in vitro system using Col E3 plasmid DNA, as a template, confirming the previous notion that the structural gene for colicin E3 protein resides on the Col E3 plasmid.