Genetic studies of chromosome arm 9p have indicated the presence of one or more tumor suppressor genes involved in genetic susceptibility to various types of human cancers. To define the extent of the specific deletion of 9p21-22 in human breast cancer, we have analyzed loss of heterozygosity and homozygous deletion of 9p21-22 in 68 paired blood and tumor samples by using fluorescent multiplex polymerase chain reaction (PCR). Of these tumors, 43% (29/68), including two ductal carcinomas in situ (DCIS), showed allele loss at one or more loci analyzed. Homozygous deletion for 9p markers was detected in 7/68 (10%) of tumor samples. Eleven tumors showed allele loss at all informative loci, and 18 tumors showed selective deletion on 9p21-22. Allele deletions in six tumors did not involve microsatellite markers flanking CDKN2. The smallest common region of deletion could be defined between D9S171 and D9S126. No significant associations were observed between deletion of 9p21-22 and any of the histopathological parameters analyzed. However, the abundance of deletions of this chromosomal region still suggests that loss and inactivation of putative tumor suppressor gene(s) located on 9p21-22 may be involved in the pathogenesis of breast cancer.