The chick embryo is widely used for the study of vertebrate development, but a general, reliable loss-of-function strategy for the analysis of gene function is currently not available. By using small inhibitory hairpin RNA (siRNA) molecules generated by the mouse U6 promoter, we have applied an RNA interference approach to achieve quantitative knockdown of the neuropilin-1 (Nrp-1) receptor in chick embryos. Functional knockdown was evident in the abolition of Sema3A-induced growth cone collapse in Nrp-1-siRNA but not Nrp-2-siRNA–expressing dorsal root ganglion (DRG) neurons. Two nervous system defects in Nrp-1 mutant mice were phenocopied in embryos treated with Nrp-1 siRNA. First, DRG axons prematurely entered the dorsal horn and projected inappropriately. Second, targeted early migrating neural crest cells destined for the sympathetic chain arrested ectopically within ventral spinal nerve roots. Localized knockdown induced by specific siRNA constructs will allow rapid functional analysis of genes regulating chick neural development whilst circumventing embryonic lethal effects often associated with global gene knockout in the mouse. Developmental Dynamics 230:299–308, 2004. © 2004 Wiley-Liss, Inc.