Recent evidence supports a role for heat-shock protein 70 (hsp70) and the 26 S proteasome in regulating apoptosis, although the precise nature of their involvement is not known. In the present study, control and Bcl-x_L-overexpressing, interleukin-3-dependent FL5.12 cell lines were treated with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132). Basal proteasome activity appeared to be ≈ 30% lower in bcl-x_L cells compared with control cells using a substrate for the chymotrypsin-like activity. However, no difference in proteasome activity was detected using substrates for the trypsin-like or peptidylglutamyl peptide-hydrolysing activities. In addition, protein levels of the 20 S proteasome β-subunit, as determined by Western blot analyses, were similar in control and bcl-x_L cells, leading to the conclusion that proteasome activities were the same in these two cell lines. At 24 h after treatment with 500 nM MG132, apoptosis in bcl-x_L cells (22%) was less than that observed in control cells (34%). Concomitantly, caspase activity in control cells, as assessed by N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartyl-7-amino-4-methylcoumarin (Ac-DEVD-AMC), was twice that observed in bcl-x_L cells. By 48 h after MG132 treatment, apoptosis and caspase activity in bcl-x_Lcells were similar to those observed in control cells at 24 h. Proteasome inhibition stimulated increases in hsp70 protein levels in control and bcl-x_L cells by 12 h, although the maximal increases found in bcl-x_L cells were less. Blocking this induction with hsp70 antisense oligonucleotides potentiated apoptosis after treatment with MG132. Inhibiting caspase activity with a broad-spectrum caspase inhibitor, t-butoxycarbonyl-Asp(OMe)-fluoromethyl ketone, prevented MG132-induced apoptosis. The more specific caspase-3 inhibitor, Ac-DEVD-aldehyde, afforded less protection, although both inhibitors completely inhibited Ac-DEVD-AMC cleavage. These data indicate that both hsp70 and Bcl-x_L provide some protection against proteasome inhibitor-induced apoptosis.