A strain of Neisseria gonorrhoeae requiring arginine, proline, glutamate and cystine as nutritional supplements was transformed, in several steps, to grow in a simple mineral medium containing cystine as the only growth factor with DNA from several clinically isolated strains of this organism. Using DNA from naturally occurring auxotrophs (auxotypes) known to require arginine, hypoxanthine and uracil (Arg-Hyx-Ura-), as well as other factors, it was possible to transfer nutritional markers, one at a time, into such prototrophs to obtain seven single marker auxotrophic strains. Three different uracil markers, two different hypoxanthine markers, an arginine marker, and an isoleucine--valine markers were each introduced into separate strains. Of 114 DNA samples from independently isolated strains of N. gonorrhoeae, 54 were able to transform all seven single marker strains to prototrophy. Six of the single marker strains failed to be transformed to prototrophy by DNA samples from 43 strains, thus demonstrating that all these strains possess at least six nutritional lesions in common. Two strains were shown to contain all seven nutritional lesions, whereas several strains contained some but not all of the seven lesions. Six of the seven single marker strains have been shown to revert spontaneously to prototrophy at low frequencies. During construction of prototrophic strains it was observed that genes conferring sensitivity to growth inhibition by nutrients in complex media were occasionally transferred along with prototrophy.