Angiogenesis is essential for tumor growth and metastasis. It is regulated by numerous angiogenic factors, one of the most important being vascular endothelial growth factor (VEGF). Recently, VEGF-C, a new VEGF family member, has been identified that binds to the tyrosine kinase receptors flt-4 [VEGF receptor (VEGFR) 3] and KDR (VEGFR2). Although the importance of VEGF has been shown in many human tumor types, the contribution of VEGF-C and its primary receptor flt-4 to tumor progression is less well understood. We have therefore measured the level of VEGF-C, flt-4, and KDR mRNA by RNase protection assay and the pattern of VEGF-C expression by immunohistochemistry in 11 normal breast tissue samples and 61 invasive breast cancers. No significant difference in VEGF-C expression was observed between normal and neoplastic breast tissues (P = 0.11). There was a significant correlation between VEGF-C and both flt-4 (P = 0.02) and KDR (P = 0.0002), but no association was seen between VEGF-C and either lymph node status (P = 0.66) or number of involved nodes (P = 0.88), patient age (P = 0.83), tumor size (P = 0.20), estrogen receptor status (P = 0.67), or tumor grade (P = 0.35). No significant relationship was present between VEGF-C and vascular invasion (P = 0.30), tumor vascularity (P = 0.21), VEGF-A (P = 0.62), or thymidine phosphorylase expression (P = 1.00). VEGF-C was expressed predominantly in the cytoplasm of tumor cells, although occasional stromal components including fibroblasts were also positive. We could demonstrate no association between lymph node metastasis and either VEGF-C (P = 0.66) or flt-4 (P = 0.4). However, we did observe a significant loss of the long but not the short isoform of flt-4 in tumors compared with normal tissues (P = 0.02 and P = 0.25, respectively), and this difference was largely accounted for by the reduction of long flt-4 in node-positive tumors. These findings strongly support a role for VEGF-C/flt-4 signaling in tumor growth by enhancement of angiogenesis and/or lymphangiogenesis and suggest that differential regulation of these processes may be controlled via flt-4 isoform transcription. They further suggest that the measurement of flt-4 isoform expression may identify a patient group that is likely to have node-positive disease and therefore benefit from additional treatment and also emphasize an additional ligand interaction that could be exploited by anti-VEGFR therapy.