Abstract: Two core small nucleolar RNP (snoRNP) proteins, Nop1p (fibrillarin in vertebrates) and Nop58p (also known as Nop5p) have previously been reported to be specifically associated with the box C+D class of small nucleolar RNAs (snoRNAs). Here we report that Nop56p, a protein related in sequence to Nop58p, is a bona fide box C+D snoRNP component; all tested box C+D snoRNAs were coprecipitated with protein A-tagged Nop56p. Analysis of in vivo snoRNP assembly indicated that Nop56p was stably associated with the snoRNAs only in the presence of Nop1p. In contrast, Nop58p and Nop1p associate independently with the snoRNAs. Genetic depletion of Nop56p resulted in inhibition of early pre-rRNA processing events at sites A(0), A(1), and A(2) and mild depletion of 18S rRNA. However, Nop56p depletion did not lead to codepletion of the box C+D snoRNAs. This is in contrast to Nop58p, which was required for the accumulation of all tested box C+D snoRNAs. Unexpectedly, we found that Nop1p was specifically required for the synthesis and accumulation of box C+D snoRNAs processed from pre-mRNA introns and polycistronic transcripts.