Sera from 10 subjects in the third trimester of pregnancy and from 10 nonpregnant women were studied to elucidate the mechanism underlying decreased theophylline protein binding during pregnancy. Consistent with the physiologic hypoalbuminemia of pregnancy, serum albumin concentrations averaged only 3.2 ± 0.3 gm/dl (± SD) in pregnant subjects, compared with 4.4 ± 0.3 gm/dl in control subjects (p<1 × 10−6), and this was the main cause of decreased theophylline binding. Saturation binding studies indicated a single class of theophylline binding sites. Theophylline binding capacity (N) was greater in pregnant (N = 4.3 ± 1.0) than in nonpregnant (N = 3.3 ± 0.4) subjects, but binding affinity (Ka) averaged only 227 ± 69 (mol/L)−1in pregnant subjects, compared with 303 ± 44 (mol/L)−1in control subjects (F2,17= 4.26;p= 0.032). At a theophylline plasma concentration of 10 μg/ml, the combined effects of hypoalbuminemia and loweredKawould reduce theophylline binding to 31% ± 3% in pregnant women, compared to 39% ± 3% in nonpregnant control subjects (p<1 × 10−5). Nonesterified fatty acid concentrations were similar in both subject groups and did not contribute to the pregnancy‐associated decrease in theophylline binding.Clinical Pharmacology and Therapeutics(1990)47,68–72;