Alterations in the p14ARF tumor suppressor are frequent in many human cancers and are associated with susceptibility to melanoma, pancreatic cancer, and nervous system tumors. In addition to its p53-regulatory functions, p14ARF has been shown to influence ribosome biogenesis and to regulate the endoribonuclease B23, but there remains considerable controversy about its nucleolar role. We sought to clarify the activities of p14 ARF by studying its interaction with ribosomes. We show that p14 ARF and B23 interact within the nucleolar 60 S preribosomal particle and that this interaction does not require rRNA. In contrast to previous reports, we found that expression of p14ARF does not significantly alter ribosome biogenesis but inhibits polysome formation and protein translation in vivo. These results suggest a ribosome-dependent p14 ARF pathway that regulates cell growth and thus complements p53-dependent p14ARF functions.