Peritumoral emboli assessed on hematoxylin-eosin-stained slides are taken into account for treatment of patients with operable breast cancer. We assessed whether immunostaining with D2-40 improves the prognostic significance of emboli in a group of tumors with a large immunohistochemical sampling and a long-term follow-up. Topography, number, and extension of hematoxylin-eosin and D2-40 emboli were compared in 94 node-negative breast cancers (median number of immunostained slides per tumor: 3). Metastasis-free survival of patients with or without hematoxylin-eosin and/or D2-40 emboli were evaluated (median follow-up of 178 months). Hematoxylin-eosin emboli were detected in 14 (15%) tumors and were located at distance from the tumor. D2-40 emboli were detected in 39 (41%) tumors and was often multiple (n=30), extensive (n=23), located within (n=13), close to (n=10) or at distance from the tumor (n=16). The 12 distant hematoxylin-eosin and D2-40 emboli were located in the same vessels (seven missed at the first hematoxylin-eosin examination and secondarily diagnosed by D2-40 staining). A difference in metastasis-free survival was found only between patients with no D2-40 emboli and those with distant D2-40 emboli (P=0.02). D2-40 emboli located within or close to the tumor had no prognostic value. Comparing the metastasis-free survival of patients with or without hematoxylin-eosin emboli, the prognostically unfavorable significance of hematoxylin-eosin emboli was improved when taking into account the seven patients with missed emboli at the first examination and secondarily diagnosed by D2-40 staining (P=0.006 vs 0.003). To conclude, D2-40 increases the diagnostic sensitivity of emboli in breast carcinoma and the high incidence of D2-40 emboli might be related to the number of immunostained slides per case. Nevertheless, only distant D2-40+ emboli had a prognostic impact. In practice, D2-40 might be useful to detect missed hematoxylin-eosin emboli especially in cases without any other prognostically unfavorable criterion.