The present study examined the protein binding of a novel antiestrogen, Analog II, in rat serum and amniotic fluid. Binding experiments were conducted both in vitro and in vivo using a tritiated derivative of Analog II to measure total protein binding. Free and bound drugs were separated using the dextran-coated charcoal method. It was determined that 80-90% of Analog II in the circulation is bound to plasma protein in both mature and immature rats. The in vitro study indicated that the binding of Analog II to protein is nonspecific except in amniotic fluid and serum from 10-day old rats, where Analog II binding could be partially reversed by the addition of tamoxifen. The results suggest that Analog II binds to a specific triphenylethylene-associated binding site on neonatal rat serum and amniotic protein.