Objective: The purpose of this study was to analyze the incidence of early-onset neonatal sepsis and the presence of antibiotic resistance of the isolated bacteria and its relationship to antibiotic chemoprophylaxis that occurred during the 4 years that followed the publication of the most recent group B streptococcal guidelines. Study Design: A prospective cohort study was performed between January 1, 1997, and December 31, 2000. All cases of early-onset neonatal sepsis were gathered prospectively. Data concerning the use of antepartum antibiotics, the isolated bacteria, and the presence of drug resistance were collected. These data were analyzed separately and were combined with published data from the preceding 6 years. Results: A total of 26 cases of blood culture-proved early-onset neonatal sepsis occurred during the current 4-year study period. Group B Streptococcus was responsible for 10 cases, and the remaining 16 cases were non-group B streptococcal organisms. Of these 16 cases, 11 parturients received antibiotic chemoprophylaxis, and 10 of the isolates (91%) were resistant to the drug that was administered, compared with only one resistant bacteria (20%) in the 5 parturients who did not receive treatment (P =.01). In combining these 16 cases with the cases from the preceding 6 years, a total of 43 cases of non-group B streptococcal sepsis occurred in 49,788 deliveries. Of these, 26 mothers were given antepartum antibiotics, and 23 of the bacterial isolates (88%) exhibited resistance, compared with only 3 of the 17 cases (18%) in which antibiotics were not dispensed (P <.00001). However, because the overall use of antepartum antibiotics increased over time, the attack rate for early-onset group B Streptococcus significantly decreased by 75% (P <.000001). Conclusion: When early-onset neonatal sepsis develops in a case in which antepartum chemoprophylaxis was used, the bacterial isolate will most likely demonstrate resistance to the antibiotic that was administered. However, the development of early-onset group B streptococcal neonatal sepsis significantly decreased as the use of antepartum antibiotics increased. Thus, the number of prevented infections from antepartum antibiotic use may still outweigh the problems that are seen when resistant bacterial infections arise. Nevertheless, based on the current protocols, a large number of parturients are candidates for antibiotic chemoprophylaxis and this, in conjunction with the global concern of bacterial drug resistance, should be motivation to examine alternative methods, such as vaginal washing or immunotherapy, for decreasing infection. (Am J Obstet Gynecol 2002;187:495-500.)