The Saccharomyces cerevisiae gene RRP1 encodes an essential, evolutionarily conserved protein necessary for biogenesis of 60S ribosomal subunits. Processing of 27S pre-ribosomal RNA to mature 25S rRNA is blocked and 60S subunits are deficient in the temperature-sensitive rrp1-1 mutant. We have used recent advances in proteomic analysis to examine in more detail the function of Rrp1p in ribosome biogenesis. We show that Rrp1p is a nucleolar protein associated with several distinct 66S pre-ribosomal particles. These pre-ribosomes contain ribosomal proteins plus at least 28 nonribosomal proteins necessary for production of 60S ribosomal subunits. Inactivation of Rrp1p inhibits processing of 27SA(3) to 27SB(S) pre-rRNA and of 27SB pre-rRNA to 7S plus 25.5S pre-rRNA. Thus, in the rrp1-1 mutant, 66S pre-ribosomal particles accumulate that contain 27SA(3) and 27SB(L) pre-ribosomal RNAs.