Objective: It is uncertain whether neonatal infection with hepatitis B, despite treatment after delivery with immunoglobulin and vaccine, is the result of prior in utero transmission of the virus or treatment failure. Furthermore, the potential risk of hepatitis B transmission from the mother to the fetus at the time a genetic amniocentesis is performed is also a concern. In an attempt to better elucidate these controversies, amniotic fluid and cord blood specimens obtained from pregnant women positive for hepatitis B surface antigen were analyzed for the presence of hepatitis B surface antigen and hepatitis B deoxyribonucleic acid. Study Design: This study was a prospective longitudinal analysis that identified hepatitis B surface antigen–positive patients who presented for amniocentesis. Cord blood was obtained from these patients at the time of delivery. Cord blood was also obtained from a group of hepatitis B surface antigen–positive patients for whom no amniocentesis was performed. All samples were analyzed for the presence of hepatitis B surface antigen and hepatitis B deoxyribonucleic acid. Results: A total of 121 hepatitis B surface antigen–positive pregnant women were identified. In the 72 pregnancies in which amniocentesis was not performed, 18% of the cord blood samples were positive for hepatitis B surface antigen and 4% were positive for hepatitis B deoxyribonucleic acid. Of 47 amniocentesis fluid samples, 32% were positive for hepatitis B surface antigen but all were negative for hepatitis B virus deoxyribonucleic acid. Of 30 cord blood samples from patients who underwent an amniocentesis, 27% were positive for hepatitis B surface antigen, but all were negative for hepatitis B virus deoxyribonucleic acid. Conclusions: This study found that hepatitis B viral deoxyribonucleic acid is rarely present in cord blood and was not identified in amniotic fluid obtained by amniocentesis. This finding suggests that in utero transmission of the virus is rare prior to the onset of labor. These data further confirm the reports in the current literature that the risk of hepatitis B transmission to the fetus during amniocentesis is low. Because hepatitis B surface antigen can exist as an isolated entity devoid of nuclear material, in some cases this protein may be able to traverse the placental and amniotic membrane barrier in a manner similar to other proteins, such as ⍺-fetoprotein. Recommendations for genetic amniocentesis in women positive for hepatitis B surface antigen are discussed. (Am J Obstet Gynecol 2001;184:1514-20.)