Synthesis of mRNA and rRNA occur in the chromatin-rich nucleoplasm and the nucleolus, respectively. Nevertheless, we here report that a Saccharomyces cerevisiae gene, MTX3, previously implicated in mRNA transport, codes for a novel essential 28-kDa nucleolar protein. Moreover, in mtr3-1 the accumulated polyA(+) RNA actually colocalizes with nucleolar antigens, the nucleolus becomes somewhat disorganized, and rRNA synthesis and processing are inhibited. A strain with a ts conditional mutation in RNA polymerase I also shows nucleolar accumulation of polyA(+) RNA, whereas strains with mutations in the nucleolar protein Nop1p do not. Thus, in several mutant backgrounds, when mRNA cannot be exported it concentrates in the nucleolus. mRNA may normally encounter nucleolar components before export and proteins such as Mtr3p may be critical for export of both mRNA and ribosomal subunits.