Ebp2 has an essential role in the biogenesis of 60S ribosomal subunits. Synthetic-sick alleles with the ebp2-14 mutation were screened. The mutations were localized to the ARD1 and NAT1 genes, which encode the catalytic subunit and the auxiliary subunit of N(α)-acetyltransferase NatA respectively. Polysome analyses revealed that ard1Δ and nat1Δ caused a synergistic defect with ebp2-14 in the assembly of 60S ribosomal subunits. To identify the proteins that functionally interact with NatA, we designed mutants in which the second amino acid was substituted for proline in Ebp2 and functionally related proteins: Brx1, a partner of Ebp2 in ribosome biogenesis, and the ribosomal protein L36a/b, overexpression of which suppresses a growth defect in ebp2-14. Among these, only brx1-S2P exhibited a synthetic defect with ebp2-14. These results suggest that optimal NatA function is important to the cooperative function of Brx1 with Ebp2 in 60S ribosomal subunit biogenesis.