Nucleotide sequence analysis of the ERBB2 proton-oncogene in human breast tumors revealed a sequence variation at codons 654 and 655, corresponding to the transmembrane region of the protein. To investigate this polymorphism, genotyping of 340 unrelated individuals was conducted by allele-specific oligonucleotide (ASO) hybridization of DNA amplified from the ERBB2 transmembrane region. ASO hybridization under competition conditions allowed rapid and unambiguous assignment of alleles and revealed the occurrence of a new allele, B3, encoding Val-Val. The sensitivity and the specificity of the ASO competition hybridization technique indicate that the method is generally applicable to the analysis of polymorphisms in the human genome.