[3H]17 alpha-Ethinylestradiol [( 3H]EE2; 5 micrograms/kg, 98.5 muCi) was administered to a female rat. After 18 hr less than 0.02% of the dose was present per ml plasma. Approximately 60% of radioactivity present in plasma was irreversibly bound to proteins, as determined by exhaustive solvent extraction and by high performance ion exchange chromatography of proteins after removal of unbound metabolites with activated charcoal. After chronic administration of [3H]EE2 (5 micrograms/kg; 2 muCi per day) for 22 days, there was a three- to fourfold accumulation of radioactivity in the plasma, together with an accumulation of radioactivity in the lung, liver, kidney, spleen and brain, compared to animals receiving a single dose. The spleen showed the greatest (greater than tenfold) significant (P less than 0.001) accumulation of radioactivity. There was a greater increase in radioactivity irreversibly bound to the soluble fraction than to the microsomal fraction of the liver. [3H]EE2 was conjugated to rat serum proteins by incubation with rat microsomes in vitro. Upon administration to female rats, the [3H]EE2-rat serum protein conjugate had a small volume of distribution (12.5 +/- 0.5 ml), and its plasma concentration declined slowly (t 1/2 = 450 +/- 140 min). Immunization of male New Zealand White rabbits with a chemically synthesized conjugate of 2-hydroxyethinylestradiol (2-OH-EE2) and human serum albumin produced antibodies which bound EE2 and 2-OH-EE2 but not estrone. These data indicate that although reactive metabolite formation represents a minor biotransformation, drug protein conjugates may accumulate during chronic administration.