Human epidermal growth factor receptor-2 (HER2) is a well-recognized growth-promoting factor in cancer, although its application to urothelial carcinoma has been limited because of a low frequency of gene amplification. We evaluated HER2 protein expression and gene amplification in micropapillary carcinoma, a rare but highly aggressive variant of urothelial carcinoma by dual-color in situ hybridization. Gene amplification was defined by a HER2:CHR17 ratio of ≥2.2; low and high levels of amplification were further defined as <2.5 and ≥2.5, respectively. Immunohistochemistry was used to determine HER2 protein expression using the American Society of Clinical Oncology/College of American Pathologists Guidelines of HER2 staining. Protein expression, gene amplification, and chromosome 17 aneusomy were compared by Jonchkeere-Terpstra and Cochran-Armitage trend tests. In all, 19 of the 20 micropapillary carcinoma samples yielded usable dual-color in situ hybridization and immunohistochemistry results for evaluation. Overall, 68% (n=13) demonstrated HER2 protein expression of 2+ to 3+ staining. Gene amplification was present in 42% of samples (n=8), with 100% correlation with 2+ and 3+ protein expression. Gene amplification and protein expression were significantly associated (P=0.01). Overall, 53% of samples (n=10) had aneusomy of chromosome 17. Chromosome 17 aneusomy was present in approximately half of the samples evaluated, suggesting inherent genomic instability in this variant of urothelial carcinoma. However, increased HER2:CHR17 ratios demonstrate increased HER2 expression due to amplification in the majority of micropapillary carcinomas. These results suggest that HER2-targeted therapy may be successful on the genomic level in patients with this disease.