In primary cultures of human neurons, 17beta-estradiol (17beta-E2) prevents caspase-6-mediated cell death and induces a caspase inhibitory factor (CIF) inhibiting active caspase-6 (Csp-6) in vitro. Here, we show that treatment of neurons with 17beta-E2 results in a proteasomal-dependent but ubiquitin-independent degradation of endogenous and exogenous active Csp-6 in live neurons and in cell free assays, respectively. We further show that the proteasomal-dependent degradation of Csp-6 is not required for its inhibition. Using several protease inhibitors, we find that leupeptin, E-64, and ALLN prevent inhibition of recombinant active Csp-6 (R-Csp-6) in 17beta-E2-treated neuronal protein extracts. Because all three protease inhibitors have the ability to inhibit cysteine proteases, we believe that a cysteinyl protease activity may be required for 17beta-E2-mediated inhibition of active Csp-6. However, we exclude caspases, calpains, and cathepsins as potential cysteinyl proteases involved in the 17beta-E2-mediated Csp-6 inhibition. The results suggest that a proteolytic activity inhibited by leupeptin, E-64, and ALLN is needed to inhibit Csp-6 and that the inhibited Csp-6 is subsequently degraded by the proteasome. The mechanism of 17beta-E2-mediated inhibition of Csp-6 is different from the ubiquitin-dependent proteasomal degradation of Csp-3 and Csp-7 by XIAP and cIAP2 but consistent with the mechanism of Baculovirus p35 inhibition of caspases.