The administration of ampicillin to three women in the last trimester of pregnancy caused a transient reduction in the plasma concentration of total conjugated estriol and of estriol-16alpha-glucuronide, as measured by a specific radioimmunoassay. Similar changes were seen in plasma conjugated estrone and estradiol levels, but the effect seemed to be more prolonged. In most cases, the reduction in plasma conjugated estrogens was greatest on the second and third day of ampicillin administration, the pattern being similar to that described previously for urinary estriol and other estrogens. There were no obvious differences in the ampicillin effect between the morning and afternoon plasma levels. Ampicillin caused no consistent change in the plasma levels of unconjugated estrone, estradiol, and estriol or in plasma progesterone. These results are discussed with consideration of our present understanding of the effect of ampicillin on the intestinal metabolism and enterohepatic circulation of steroids.