TP53 is the most commonly mutated tumor suppressor gene in human cancers. The amplification and overexpression of HDM2 plays a role in tumorigenesis via inactivation of p53-dependent cell cycle arrest. p14^ARF, an alternate transcript of the INK4A tumor suppressor locus, prevents hdm2-induced transcriptional silencing of p53 by binding hdm2. The role of this p14^ARF-hdm2-p53 regulatory pathway in breast carcinoma is unknown. We hypothesized that p14 ^ARF mutations and HDM2 gene amplification may be alternative mechanisms of p53 inactivation in breast cancer. Mutational analysis of TP53 (exons 5–9) and exon 1β of p14 ^ARF was performed by PCR-SSCP and putative mutations were confirmed by sequencing. p14^ARF mRNA expression was evaluated by RT-PCR and the presence of HDM2 gene amplification by differential PCR. Among the cell lines, 7/14 (50%) harbored TP53 mutations and 2/14 (14%) had a deletion of p14 ^ARF exon 1β with no detectable p14^ARF mRNA. None demonstrated HDM2 gene amplification. TP53 mutations were identified in 7/36 (19%) breast tumors and HDM2 amplification in 2/30 (7%) tumors. All the tumors contained an intact p14 ^ARF exon 1β with corresponding expression of the mRNA. Alterations in the various components of this regulatory pathway were identified in nine (64%) cell lines and 25% of the 36 breast cancers with TP53 mutation being the predominant aberration. Although p14 ^ARF mutations and HDM2 gene amplification appear to be uncommon events in breast carcinoma, deregulation of this pathway may occur via alternative mechanisms in breast carcinogenesis.