OBJECTIVE: Intrapartum penicillin G prophylaxis aims to prevent early-onset group B streptococci (GBS) sepsis by interrupting vertical transmission. We examined the relationship between duration of prophylaxis and fetal serum penicillin G levels among fetuses exposed to fewer than 4 hours of prophylaxis compared with longer durations. METHODS: In this prospective cohort study, 98 laboring GBS-positive women carrying singleton gestations at 37 weeks or greater were administered 5 million units of intravenous penicillin G followed by 2.5 million units every 4 hours until delivery. Umbilical cord blood samples were collected at delivery, and penicillin G levels were measured by high-performance liquid chromatography. Intraassay and interassay coefficients of variation were less than 3%. RESULTS: Fetuses exposed to fewer than 4 hours of prophylaxis had higher penicillin G levels than those exposed to greater than 4 hours (P=.003). In multivariable linear regression analysis, fetal penicillin G levels were determined by duration of exposure, time since last dose, dosage, and number of doses, but not maternal body mass index. Penicillin G levels increased linearly until 1 hour (R(2)=.40) and then decreased rapidly according to a power-decay model (R(2)=.67). All subgroups analyzed were above the minimal inhibitory concentration (MIC) for GBS (0.1 micrograms/mL)(P<.002). Individual samples were 10-179-fold above the MIC. In patients receiving maintenance dosing, penicillin G did not accumulate in the cord blood and returned to baseline after each 4-hour interval. CONCLUSION: Short durations of prophylaxis achieved levels significantly above the MIC, suggesting a benefit even in precipitous labors. The designation of infants exposed to fewer than 4 hours of prophylaxis as particularly at risk for GBS sepsis may be pharmacokinetically inaccurate.