The tumor suppressor p16(INK4a) has been shown to be inactivated in numerous cancer lines and primary tumors. Recently, we reported loss of heterozygosity of the region in which p16(INK4a) is located in more than one-half of primary breast tumors. However, mutational analysis of these same tumors revealed mutation of p16(INK4a) to be infrequent. Other possible modes of inactivation, such as de novo methylation and homozygous deletion, have since been shown to occur in numerous neoplasias. Furthering the complexity of this locus, a transcript overlapping the p16(INK4a) coding sequence and encoding a novel peptide with growth-suppressive activity, p19(ARF), has been described. To clearly elucidate the target of aberrations affecting this subchromosomal region and approximate frequency in breast cancer, we performed a comprehensive study including p16 deletion analysis by means of interphase chromosomal fluorescence in situ hybridization, methylation analysis of the first exon encoding p16(INK4a) (exon 1alpha), mutational analysis of exon 1beta by single-strand conformational polymorphism analysis of p19(ARF) transcripts, and expression of both alpha and beta transcripts by reverse transcription PCR. Homozygous deletion of p16, as determined by interphase chromosomal fluorescence in situ hybridization, was observed in 3 of 18 (17%) tumors analyzed, whereas de novo methylation of exon 1alpha was observed in an additional 17% (4 of 23). Reduced expression of p16(INK4a) was observed in 11 tumors (48%), including all those in which homozygous deletion or complete methylation was observed. No mutations of exon 1 beta were detected, and expression of its transcript was variable, with 13% demonstrating decreased expression and 17% demonstrating overexpression. These results further support p16(INK4a) as a target of inactivation in the 9p21 region for breast cancer.