To investigate the distribution of penicillin in the maternal-placental-fetal unit at term gestation.Twenty-five healthy gravidas at 38-39 weeks' gestation scheduled for elective repeat cesarean delivery under spinal anesthesia received benzathine penicillin G, 2.4 million units intramuscularly (IM) preoperatively. Ten women delivered 1 day after injection, five delivered 2-3 days after, and ten delivered 7 days after. We collected maternal serum and cerebrospinal fluid, amniotic fluid (AF), and cord serum at delivery. Penicillin levels were measured using a validated agar disc diffusion method (sensitivity 0.006 micrograms/mL) with Micrococcus lutea as the test organism.There was no significant difference in mean penicillin levels at day 1, day 2-3, or day 7 for maternal serum, maternal cerebrospinal fluid, cord serum, or AF. The mean (+/- standard error) penicillin concentration (range 0.005-0.59 micrograms/mL) in maternal serum declined from 0.14 +/- 0.04 micrograms/mL 1 day after injection to 0.08 +/- 0.06 micrograms/mL 7 days after injection. The proportion of patients with a penicillin concentration at or above 0.018 micrograms/mL in the maternal serum declined significantly from day 1 to day 7 (P = .03). Overall, nine of 25 women (36%) had serum penicillin levels that were less than 0.018 micrograms/mL.A wide range of penicillin levels were observed in gravidas at term in the maternal serum, cerebrospinal fluid, umbilical cord serum, and AF within 1 week after 2.4 million units of benzathine penicillin G IM. We speculate that altered pharmacokinetics may affect the efficacy of this drug for prevention of congenital syphilis in the near-term gestation.