Several benzoquinoid ansamycins, e.g., herbimycin A and geldanamycin, have been widely used as inhibitors of tyrosine kinases. We recently reported that exposure to herbimycin A and several analogs depletes the erbB2 gene product p185 in human breast cancer cells. In order to explore the mechanism of this specific degradation of p185, a biologically active ansamycin incorporating a photoaffinity label was synthesized. This compound, CP202509, specifically bound to a 100 kD protein (p100) in intact SKBr3 cells and in fibroblasts transfected with the c-erbB2 or v-src oncogenes. Binding of other ansamycin analogs to p100, as measured indirectly by their ability to inhibit CP202509 binding, correlated with their ability to lower p185 protein and phosphotyrosine in SKBr3 cells. These results suggest that the ansamycins may deplete tyrosine kinases through binding to this protein.