Ubiquitination is a key event for protein degradation by the proteasome system, membrane protein internalization, and protein trafficking among cellular compartments. Few data are available on the role of the ubiquitin-proteasome system (UPS) in the trafficking of neuronal nicotinic acetylcholine receptors (nAChRs). Experiments conducted in neuron-like differentiated rat pheochromocytoma cells (PC12 cells) show that the alpha3, beta2, and beta4 nAChR subunits are ubiquitinated and that their ubiquitination is necessary for degradation. A 24-h treatment with the proteasome inhibitor PS-341 increased the total levels of alpha3 and the two beta subunits in both whole cell lysates and fractions enriched for the ER/Golgi compartment. nAChR subunit upregulation was also detected in plasma membrane-enriched fractions. Inhibition of the lysosomal degradation machinery by E-64 had a significantly smaller effect on nAChR turnover. The present data, together with previous results showing that the alpha7 nAChR subunit is a target of the UPS, point to a prominent role of the proteasome in nAChR trafficking.