The profile of catechol estrogen formation by pig conceptuses from d 14 to 20 of pregnancy (d 0 = 1st d of estrus) was studied using a direct product isolation assay for estrogen-2/4-hydroxylase (E-2/4-H). High performance liquid chromatography was coupled to detection of radioactive products in a flowing system to separate the 2- and 4-hydroxyestradiol (2- and 4-OH-E2) formed from estradiol. The major product was 2-OH-E2; its formation increased with increasing amounts of nicotinamide cofactors and exhibited a preference for NADPH. Maximum velocities were 909 and 154 pmol.mg protein-1.30 min-1, and apparent Michaelis constants were 7.14 and 5.12 microns, for 2- and 4-OH-E2 formation, respectively. Homogenates of d-13 conceptuses produced 1,151 +/- 142 pmol.mg protein-1.30 min-1 of 2-OH-E2. Estradiol-2-hydroxylase was affected (P less than .01) by day of pregnancy, and day effects from d 14 to 20 were described by a fourth-order equation (P less than .001). Conceptuses collected on d 14 produced 119 +/- 15.4 pmol of 2-OH-E2.mg protein-1.30 min-1. By d 15, E-2-activity had declined to 15.7 +/- 1.8 pmol.mg protein-1.30 min-1. Formation of 2-OH-E2 increased to 64.2 +/- 15.4 pmol.mg protein-1.30 min-1 on d 17, remained stable through d 19, and was 39.4 +/- 13.8 pmol.mg protein-1. 30 min-1 on d 20. Therefore, E-2/4-H activity appears to be regulated from d 14 to 20 of pregnancy in pigs; it may participate in the establishment of pregnancy.