A system of auxotyping described in 1973 is based on the differing nutritional requirement patterns of Neisseria gonorrhoeae strains. Our ongoing evaluation of the reliability of auxotyping has involved a study of the constancy of characteristics of gonococci isolated at one time from two or more sites of a given subject. The auxotypes and minimal inhibitory concentration (MIC) of penicillin G were determined for 181 isolates obtained from 84 patients with uncomplicated gonorrhea, for 16 isolates from 8 couples with uncomplicated gonorrhea, and for 21 isolates from 12 other patients, 9 with disseminated gonococcal infection and three consorts. The penicillin MIC served to distinguish between many members of auxotypes 1, 2, and 3, which are commonly involved in uncomplicated gonorrhea. Thus, for proline-requiring gonococci (auxotype 2) the MIC ranged from 0.01 to 1.2 IU of penicillin per ml. The profile of gonococcal responses to seven other antibacterial drugs provided useful additional information where the extent of phenotypic similarity was in doubt. In all but seven instances, the gonococci isolated from different sites of the same patient, or from a consort, had the same nutritional requirements and penicillin MIC. The gonococci isolated from one patient with disseminated gonococcal infection and from one of her two sexual contacts had nutritional requirements for arginine, hypoxanthine, uracil, and thiamine pyrophosphate, whereas the strain isolated from her second contact differed in having no requirement for thiamine pyrophosphate. The paired cervical and rectal isolates from one patient with uncomplicated gonorrhea differed only with respect to a requirement for hypoxanthine. Pairs of isolates from three patients differed slightly in degree of susceptibility to penicillin. In the remaining two instances, however, numerous differences between the isolates from the endocervix and the anal canal of a given patient indicated the presence of concomitant infections with different strains of N. gonorrhoeae.