N-acetyl-L-leucyl-L-leucyl-L-norleucinal, (LLnL), which inhibits proteasomes in addition to other proteases, was found to prolong the association of major histocompatibility complex class I molecules with the transporters associated with antigen processing (TAP), and to slow their transport out of the endoplasmic reticulum (ER). LLnL induced a reversible accumulation of ubiquitinated proteins and changed the spectrum of peptides bound by class I molecules. These effects can probably be attributed to proteasome inhibition. Unexpectedly, in the TAP-deficient cell line .174, the rate of intracellular transport of human histocompatibility leukocyte antigen (HLA) A2 was also reduced by LLnL, and the generation of most HLA-A2-associated signal sequence peptides was inhibited. The inhibition of HLA-A2 transport in .174 cells was found to be less sensitive to LLnL than in wild-type cells, and a similar difference was found for a second protease inhibitor, benzyloxycarbonyl-L-leucyl-L-leucyl-L-phenylalanilal. These data suggest that under some conditions such inhibitors can block trimming of peptides by an ER peptidase in addition to inhibiting cytosolic peptide generation.