OBJECTIVE To estimate compliance with the 2002 revised perinatal group B streptococci (GBS) prevention guidelines in Tennessee, which recommend universal GBS screening of pregnant women at 35–37 weeks of gestation and, when indicated, administration of intrapartum chemoprophylaxis. METHODS Active Bacterial Core surveillance conducts active, population-based surveillance for invasive GBS disease in 11 Tennessee counties. A retrospective case–cohort study was conducted using a stratified random sample of all live births in surveillance hospitals during 2003–2004, including all early-onset GBS cases. Factors associated with GBS screening and lack of optimal GBS chemoprophylaxis were analyzed using logistic regression. RESULTS Screening was performed for 84.7% of pregnant women, but 26.3% of prenatal tests with documented test dates were performed before 35 weeks of gestation. Among women with an indication for GBS prophylaxis, 61.2% received optimal chemoprophylaxis, defined as initiation of a recommended antibiotic 4 hours or more before delivery. When the analysis was restricted to women who were admitted 4 hours or more before delivery, 70.9% received optimal chemoprophylaxis. Women not receiving optimal chemoprophylaxis were more likely to have penicillin allergy (11.7% compared with 2.5%, adjusted odds ratio [OR] 8.58, 95% confidence interval [CI] 1.57–47.04) or preterm delivery (45.5% compared with 13.2%, adjusted OR 5.52, 95% CI 2.29–13.30) and were less likely to have received the recommended prenatal serologic testing for other infectious diseases (77.9% compared with 91.1%, adjusted OR 0.30, 95% CI 0.09–0.98). Forty cases of early-onset GBS were identified (0.36 per 1,000 live births); 25% of these neonates were born to women who received screening at 35 weeks of gestation or later and, when indicated, optimal chemoprophylaxis. CONCLUSION Universal prenatal GBS screening was implemented widely in Tennessee, although the timing of screening and administration of chemoprophylaxis often were not optimal. A substantial burden of early-onset GBS disease occurs despite optimal prenatal screening and chemoprophylaxis, suggesting that alternative strategies, such as vaccination, are needed. LEVEL OF EVIDENCE II