Placental membrane anatomy was evaluated after delivery in 65 twin pregnancies undergoing genetic amniocentesis with amniotic fluid alpha-fetoprotein and acetylcholinesterase determinations when indicated. Each twin member in the 57 uncomplicated twin pregnancies was found to have an amniotic fluid alpha-fetoprotein concentration similar to that of singleton pregnancies of an equivalent gestational age. Eight twin pregnancies were found to be discordant for fetal anomalies associated with an elevated amniotic fluid alpha-fetoprotein and acetylcholinesterase level. The amniotic fluid alpha-fetoprotein concentration in the unaffected fetus in these eight cases was found to be dependent on placental membrane structure. Of the five discordant twin pregnancies with diamnionic-dichorionic placentas, all unaffected twins demonstrated an amniotic fluid alpha-fetoprotein concentration within the normal range for gestational age with a normal acetylcholinesterase level. In three twin pairs with diamnionic-monochorionic placentas, the unaffected twin member had significantly elevated amniotic fluid alpha-fetoprotein and acetylcholinesterase levels, which suggested diffusion of alpha-fetoprotein and acetylcholinesterase through the amnion-amnion interface.