The 26S proteasome complex plays an essential role in intracellular protein degradation. The 26S complex contains one 20S proteolytic core and two 19S regulatory particles. We investigated the kinetic properties of the 26S proteasome and the potential regulation by estrogen in mice liver in control conditions (ovariectomized placebo treated) and after 10 days of estrogen treatment. Livers were homogenized with a low concentration of detergent to preserve proteasome integrity (mM): 50 Tris-HCl, 250 sucrose, 5 MgCl2, 2 ATP, 1 DTT, 0.5 EDTA, and 0.025% digitonin, pH 7.5. The assay buffer contained (mM): 50 Tris-HCl, 40 KCl, 5 MgCl2, 0.5 ATP, 1 DTT, pH 7.5. Activity was measured at 37 oC using three fluorescent substrates, s1, caspase-like (Z-LLE-AMC), s2, trypsin-like (Boc-LSTR-AMC), and s5, chymotrypsin-like (Suc-LLVY-AMC). With all substrates, the proteolytic kinetics showed three phases: 1) a delay reflecting an initial rate-limiting process (binding of the peptide substrates to the 19S regulatory particles and the translocation to the proteolytic core), 2) a linear time-dependent proteolysis (degradation process in the 20S chamber), and 3) a saturation phase. Activity was measured as a function of substrate concentration (10-500μM) at constant total protein (s1, s2 100 μg; β5, 50 μg). Increasing the substrate concentration did not affect the delay phase, while it increased the degradation rate and the saturation level. Increasing the protein concentration with constant substrate s1, s2 and s5 50 μM) seemed to reduce the delay phase, while the linear activity and the saturation levels peaked at 100 μg protein. Estrogen treatment selectively stimulated proteolytic activity of the s2 subunit trypsin-like activity. We conclude that proteasome activity has at least three sequential states with selective modulation by hormones of the proteolytic activity.