Susceptibility to penicillin of 30 strains (one isolate per patient) of Neisseria meningitidis isolated from blood (N = 19) or cerebrospinal fluid (N = 11) was studied by two methods. Minimum inhibitory concentrations (MICs) obtained with the Etest were compared to those obtained by the National Committee for Clinical Laboratory Standards agar dilution method. Twenty meningococci (67%) relatively resistant to penicillin were identified by both methods. The mean MIC from the reference method was 0.32 micrograms/ml (range, 0.2-1) and by the Etest method was 0.35 micrograms/ml (range, 0.19-1.25). All MICs obtained by the Etest method were within one dilution of the MICs obtained by the reference method. Because of the increase in penicillin MIC of meningococcal isolates in Spain, we evaluated the performance of the Etest as an alternative method for penicillin-susceptibility testing of N. meningitidis. The Etest is a simple and accurate method for determining the susceptibility of N. meningitidis to penicillin.