Previous studies have suggested that the rostrocaudal patterning of branchial arches in the vertebrate embryo derives from a coordinate segmental specification of gene expression in rhombomeres (r) and neural crest. However, expression of the Krox-20 gene is restricted to neural crest cells migrating to the third branchial arch, apparently from r5, whereas this rhombomere contributes cells to both the second and third arches. We examined in the chick embryo how this spatially restricted expression is established. Expression occurs in precursors in both r5 and r6, and we show by cell labelling that both rhombomeres contribute to Krox-20-expressing neural crest, emigration occurring first from r6 and later caudally from r5. Krox-20 transcripts are not detected in some precursors in rostral r5, presaging the lack of expression in cells migrating rostrally from this rhombomere. After transposition of r6 to the position of r4 or r5, many Krox-20-expressing cells migrate rostral to the otic vesicle, whereas when r5 is transplanted to the position of r4, only a small number of migrating cells express Krox-20. These results indicate that, in the chick, Krox-20 expression in branchial neural crest does not correlate with rhombomeric segmentation, and that there may be intrinsic differences in regulation between the r5 and r6 Krox-20-expressing populations.