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The purpose of this study was to determine the endogenous concentrations of estrogens, particularly estradiol-17β (E2β), in edible tissues of beef cattle (females and intact and neutered males) and the concentrations of E2β, and trenbolone beta and alpha (βTb, αTb) after an E2β and/or trenbolone acetate (TA) ear implant. Radioimmunoassays were validated for quantitation of E2β (active isomer), E2α, estrone (E1), βTb and αTb for bovine muscle, liver, kidney and fat tissues. The criteria of accuracy, precision, specificity and sensitivity were applied according to the standards of the U.S. Food & Drug Administration. In steer tissues, endogenous E2β was <15 ppt, as was heifer muscle; but heifer liver and kidney were 3-fold greater. An E2β implant in steers had no effect on muscle E2β concentration, but increased E2β in liver and fat 4- and 3-fold, respectively, but by 24 h post-implant removal, E2β had fallen by half. Tissue E1 concentrations in cyclic females were similar to E2β, but rose many fold greater than did E2β during gestation; E2β rose 3-fold during gestation. After E2β/TA implant, steer tissues had E2β concentrations equal to (for muscle and fat) and one-half (for liver) the E2β measured in E2β implant only steers; βTb was in a low range (250–380 ppt) in muscle, liver and fat and αTb was even lower, except in liver (800–1500 ppt). An implant of TA only (no E2β) resulted in βTb and αTb concentrations 2–3-fold greater in liver, kidney and fat, but no greater in muscle than βTb in tissues of E2β/TA implant steers. In conclusion, anabolic implants in steers resulted in tissue E2β concentrations less than the FDA allowable increment and βTb in the lowest quartile (0.25) of a part per billion 30 days after implant.