The human immunodeficiency virus-1 (HIV-1) Tat protein was previously reported to compete the association of PA28 regulator with the alpha 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 alpha4 and alpha7, six beta subunits of the constitutive 20S proteasome and the interferon-gamma-inducible subunits beta2i and beta5i. 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 specific interaction with distinct proteasomal subunits.