The human immunode¢ciency virus-1 (HIV-1) Tat protein was previously reported to compete the association of PA28 regulator with the K K rings of the 20S proteasome and to inhibit its peptidase activity. However, the distinct interaction sites within the proteasome complex remained to be determined. Here we show that HIV-1 Tat binds to K K4 and K K7, six L L subunits of the constitutive 20S proteasome and the interferon-Q Qinducible subunits L L2i and L L5i. A Tat^proteasome interaction can also be demonstrated in vivo and leads to inhibition of proteasomal activity. This indicates that Tat can modulate or interfere with cellular proteasome function by speci¢c interaction with distinct proteasomal subunits. ß