Cultures of Neisseria gonorrhoeae started with type 1 colonies were examined for the presence of plasmids. The cells were grown in a biphasic system containing 8-14C-adenine, then lysed. After most of the chromosomal DNA was removed by centrifugation, the remaining lysate was added to an ethidium bromide-cesium chloride solution and centrifuged to a density equilibrium. Fractions were collected and counted. A peak of radioactivity was observed in the denser fractions (30–40) that contain the covalently closed, circular DNA plasmid molecules. Treatment of the lysates with RNase removed some of the counts at the bottom of the gradient, but it had no apparent effect on the plasmid-containing fractions. The culturing, lysing, and labeling techniques described provide the means to investigate other isolates of N. gonorrhoeae for plasmids that may possess the genetic information for bacteriocinogeny and/or or pili.