Neuropilin1 (Nrp1) and Neuropilin2 (Nrp2) are nonkinase vascular endothelial growth factor receptors (VEGFR) identified in several vertebrates, which function as coreceptors for the receptor tyrosine kinases VEGFR1 and VEGFR2. We identified four zebrafish nrp genes, nrp1a, nrp1b, nrp2a, and nrp2b, and characterized their function in vascular development. We show that all nrp genes display distinct expression patterns and that nrp1a and nrp1b are expressed in the dorsal aorta, while nrp2a and nrp2b transcripts could be detected in the region of the posterior cardinal vein. Knockdown of nrp1a, nrp1b, and nrp2a resulted in improper arteriovenous connections and irregular intersegmental vessel patterning, indicating that these Nrps function in the same process. Nrp2b knockdown also caused vessel malformations and a pericardial defect. In addition, we provide evidence that the newly identified Nrps synergistically interact with VEGF in vivo. Taken together, our results show that, in zebrafish, all four neuropilins are involved in VEGF-mediated vessel development. Developmental Dynamics 231:33–42, 2004. © 2004 Wiley-Liss, Inc.