Penicillin G and ampicillin are the recommended antibiotics for prevention of early-onset GBS disease (EOGBS). We studied the antibiograms of 111 colonizing and 8 invasive strains of GBS to penicillin G, ampicillin, clindamycin, cefazolin, gentamicin, and erythromycin using both minimal inhibitory concentration (MIC) and Kirby-Bauer (KB) methods because: 1) Widespread use of penicillin G and ampicillin has increased the likelihood of emergence of resistance; and 2) The methods used to determine antibiotic susceptibility may influence the perception of antibiotic resistance and subsequent choice of prophylactic antibiotics. GBS strains were collected prospectively between 1/1/96 and 9/30/97 as part of an NICHD sponsored study of the association between passively acquired anti-GBS antibody and the risk of EOGBS. MICs were performed using prepared test panels (Microtech Medical Systems, Aurora, CA) and KBs were done in the standard fashion using antibiotic impregnated filter paper discs. MIC results showed that all colonizing strains were sensitive (MIC ≤ 0.12 mcg/ml) or moderately sensitive (MIC = 0.25-2 mcg/ml) to penicillin G and ampicillin. These levels are easily achieved in fetal blood using current regimens. In contrast, 82% of strains were of intermediate sensitivity to ampicillin and 12% were intermediate to penicillin G using the KB method. Frank resistance was noted by at least one strain to each of the other antibiotics; all were resistant to gentamicin while 9 were resistant to erythromycin, 3 to clindamycin, and one to cefazolin by MIC. All invasive strains were sensitive to ampicillin, penicillin G, clindamycin, and cefazolin by MIC, one was intermediate to erythromycin and all were resistant to gentamicin. We conclude that: 1) Continued sensitivity of GBS to members of the penicillin family, plus occurrence of at least one resistant strain for each of the other antibiotics supports continued use of penicillin G or ampicillin in prophylactic treatment regimens against EOGBS; and 2) Use of the KB method by clinical labs could lead to the erroneous conclusion that GBS are becoming resistant to penicillin G and ampicillin. Furthermore, when beta lactam antibiotics are contraindicated, clindamycin appears to be a better alternative drug than erythromycin, because of the reduced frequency of resistance.