The colicinogenic plasmid Col Ib-P9 of an Escherichia coli colicinogenic strain has been purified by agarose gel-filtration chromatography. The plasmid elutes from the column as a single peak, well separated from chromosomal fragments. When analyzed on neutral sucrose gradients the column, purified plasmid sediments at a rate characteristic for relaxed open circular Col Ib-P9 DNA. Measurements of the plasmid molecular complexity based on renaturation kinetics are consistent with the reported molecular weight for the plasmid. DNA-DNA hybridization has been employed to demonstrate a lack of sequence homology between the plasmid and the E. coli chromosome.