Transfer of plasmid DNA from penicillinase-producing Neisseria gonorrhoeae to Escherichia coli produced relatively small colonies that became pigmented in the presence of the dye, congo red. Mutations to large, non-pigmented colonies were frequent, and were the result of a variety of alterations in these plasmids. Miniplasmid formation was the major alteration that led to non-pigmented colonies. However, deletions in any of three putative replication regions resulted in non-pigmented colonies. These altered plasmids mediated significantly less beta-lactamase production than that of the parent plasmid. A new origin of replication was discovered and mapped within the miniplasmid. This origin was responsible for: 1) high plasmid copy number, 2) stability of inheritance, and 3) the majority of beta-lactamase produced in culture. The evolution of gonococcal plasmids may be followed in E. coli, since less defective plasmids produce nonpigmented colonies on congo red agar medium.