The ribosome decoding center is rich in modified rRNA nucleotides and little is known about their effects. Here, we examine the consequences of systematically deleting eight pseudouridine and 2'-O-methylation modifications in the yeast decoding center. Loss of most modifications individually has no apparent effect on cell growth. However, deletions of 2-3 modifications in the A- and P-site regions can cause (1) reduced growth rates (approximately 15%-50% slower); (2) reduced amino acid incorporation rates (14%-24% slower); and (3) a significant deficiency in free small subunits. Negative and positive interference effects were observed, as well as strong positional influences. Notably, blocking formation of a hypermodified pseudouridine in the P region delays the onset of the final cleavage event in 18S rRNA formation ( approximately 60% slower), suggesting that modification at this site could have an important role in modulating ribosome synthesis.