Stable colonial types 1, 2, 3, and 4 were prepared from eight strains of Neisseria gonorrhoeae. Four of the strains, termed laboratory strains, had been transferred over 100 times; three strains, termed clinical strains, were transferred only three to five times after isolation from patients, and one stabilized clinical strain was transferred purposefully 30 times after isolation from a patient. Colonial types of the three categories were grown on four media containing the following agents at the level used in diagnostic media: (i) vancomycin, colistin, and nystatin; (ii) these antibiotics plus trimethoprim lactate; (iii) trimethoprim lactate alone; and (iv) a control with no antimicrobial agents. When grown on media containing the antimicrobial agents, colonial types 1, 2, and 3 of all strains showed specific and consistent changes that precluded accurate identification of the types. In general, the colonies were smaller, more dense to transmission of light, and more granular than colonies grown on control medium. More colonies showed these type changes in the clinical strains and on media containing trimethoprim lactate. Colonies of type 4 showed little or no change. The changes in colonial morphology of types 1, 2, and 3 were pronounced enough to make colony typing difficult if the antimicrobial agents, particularly trimethoprim lactate, were present in media.