Recent demonstration by us of a consistent presence of solvolyzable (presumably sulfate) conjugated corticosteroids in human plasma stimulated search for a reproducible technique for the separation of various groups of steroid conjugates. It was found that a very distinct, quick and reproducible separation of steroid glucuronides from steroid sulfates, as well as steroid monosulfates from disulfates, and monoglucuronides from diglucuronides can be achieved with the aid of high voltage (H.V.) paper electrophoresis. Runs were carried out on Whatman No. 3 MM filter paper in an Ensco H.V. electrophoresis chamber, installed in a cold room (+ 4 C). The separated steroids were located by color spot tests. Mixtures of various conjugates of C18, C19 and C21 steroids were rapidly resolved, with an excellent reproducibility. With the technique developed it was possible to separate dehydroepiandrosterone (DHEA) glucuronide from DHEA sulfate during a 90-min run in a pyridine-formic-acetic acid buffer (pH 2.2) at 2,000 v and 50 mamp by a distance of 21 cm; and tetrahydrocortisone-3-glucuronide from tetrahydrocortisol-3,21-disulfate by a distance of 32 cm. The influence of various pH's, buffers and current and potential gradients upon the effectiveness of the separation was extensively studied. No formation of artifacts during the H.V. runs was observed. All steroids separated as very compact and well-defined spots. The recoveries ranged from 85 to 98%. It is concluded that H.V. paper electrophoresis is an excellent and important addition to the armamentarium of steroid methodology.