The 26S proteasome is a multicatalytic protease complex that degrades ubiquitinated proteins in eukaryotic cells. It consists of a proteolytic core (the 20S proteasome) as well as regulatory particles, which contain six ATPase (Rpt) subunits involved in unfolding and translocation of substrates to the catalytic chamber of the 20S proteasome. In this study, we used MS to analyze the N-terminal modifications of the yeast Rpt1 subunit, which contains the N-terminal recognition sequence for N-methyltransferase. Our results revealed that following the removal of the initiation Met residue of yeast Rpt1, the N-terminal Pro residue is either unmodified, mono-methylated, or di-methylated, and that this N-methylation has not been conserved throughout evolution. In order to gain a better understanding of the possible function(s) of the Pro-Lys (PK) sequence at positions 3 and 4 of yeast Rpt1, we generated mutant strains expressing an Rpt1 allele that lacks this sequence. The absence of the PK sequence abolished N-methylation, decreased cell growth, and increased sensitivity to stress. Our data suggest that N-methylation of Rpt1 and/or its PK sequence might be important in cell growth or stress tolerance in yeast.