A series of chimeric plasmids derived from the transfer factor, beta-lactamase-encoding R factor, and cryptic plasmid of Neisseria gonorrhoeae was constructed. Two of these plasmids, each lacking a 1.9-kilobase-pair (kb) HinfI fragment, could not be mobilized by the 40-kb gonococcal transfer factor to recipient strains of N. gonorrhoeae or Escherichia coli during conjugation. The proteins encoded by both the naturally occurring and chimeric plasmids were examined with an E. coli cell-free transcription-translation system and in E. coli maxicells. Six plasmid-specific proteins were identified when DNA from a naturally occurring 7.1-kb R factor was used as template. A small protein (16,000 daltons), which is apparently not encoded by the two plasmids lacking the 1.9-kb HinfI fragment, appears to be necessary for plasmid transfer during conjugation.