Strains of Neisseria gonorrhoeae that failed to grow on Thayer-Martin (T-M) and Martin-Lewis (M-L) media accounted for 2.0% of isolates at the University of Colorado Hospital and its Venereal Disease Clinic. A total of 31 inhibited and 31 control strains were compared by agar dilution testing for their susceptibilities to 13 antimicrobial agents used for treatment or in selective media. All 62 isolates were resistant to lincomycin, colistin, nystatin, amphotericin B, trimethoprim lactate, polymyxin B, and anisomycin. Vancomycin was the inhibitory antibiotic for N. gonorrhoeae in both T-M and M-L media. The vancomycin-inhibited strains were also significantly more sensitive to penicillin and ampicillin than were the control strains (P less than 0.001). The presence of the other antibiotics in selective media did not affect the minimum inhibitory concentrations of vancomycin for gonococci. All 31 inhibited strains were sensitive to 8.0 micrograms of vancomycin per ml, and 26 of these were sensitive to 2.0 microgram/ml. Decreasing the size of inoculum of gonococci results in greater inhibition by any given concentration of vancomycin. The vancomycin-sensitive strains contained significantly more arginine- hypoxanthine-, and uracil-requiring auxotypes (28 out of 31) than did the control strains (9 out of 31). As with T-M medium, some strains of gonococci will be missed when M-L medium with 4.0 micrograms of vancomycin per ml is the only medium used for the diagnosis of gonorrhea. This may be of particular importance in the confirmation of disseminated infection with Arg- Hyx- Ura- auxotypes of N. gonorrhoeae when cultures of blood, joint fluid, or skin lesions are negative.