The protein binding characteristics of the nonsteroidal aromatase inhibitor letrozole were determined using 14C-labeled letrozole. The binding of letrozole in human serum was 60.1 +/- 2.9% as a mean obtained in six individual sera and was similar in human plasma. The binding in human serum remained constant at concentrations of letrozole ranging from 10 to 500 ng/mL. A similar binding value in human serum was obtained using equilibrium dialysis and ultrafiltration technique. Albumin (binding 55.1 +/- 1.4%) is the main protein involved in the drug binding to plasma proteins. Increases in letrozole concentration (10-500 ng/mL) had no effect on binding. Albumin binding appeared to be nonsaturable with a binding capacity of 2 L/mmol. Binding to alpha1-acid glycoprotein and to gamma globulins was lower than 10%. The fraction in erythrocytes with a hematocrit of 0.4 was found to be 35.2 +/- 2.7%. Letrozole binding to serum proteins of rat, dog, mouse, and rabbit was approximately 10% lower than that in human serum and approximately 20% lower than that in baboons. Tamoxifen (100-1,500 ng/mL) had no effect on the in vitro binding of letrozole. Ex vivo binding in plasma from patients after repeated administration of letrozole alone (61.4 +/- 2.6%) was the same as after combined administration of letrozole and tamoxifen (60.0 +/- 3.2%).