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Objectives: To examine the assumption that amniotic fluid alpha-fetoprotein (AFAFP) levels are different in female and male twin fetuses. Design: Amniotic fluid levels of AFP in pregnancies with female and male fetuses in gender-concordant and gender-discordant twins were compared. A t test of p < 0.05 was considered significant. Material and Methods: Between 1995 and 1999, 332 genetic amniocenteses on twin pregnancies were performed at Meir Hospital, Kfar Saba, and Rambam Hospital, Haifa, Israel. One hundred and sixty-six were concordant for gender (84 females and 82 males) while 166 pairs differed in their gender. The amniotic fluid AFP levels of each sac were measured using fluorescent immunoassay methods by an AutoDELFIA machine. Results: The mean levels of AFAFP were lower in female twins compared to their male counterparts in same-gender twins (p = 0.07), although the difference was quite small. Nevertheless, there was no such difference between AFAFP of male versus female fetuses in gender-discordant twins. Conclusions: The levels of AFAFP were higher in the male twins of gender-concordant twins in comparison to female twins. No such difference was found between female versus male fetuses in gender-disconcordant twins.