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The zinc-finger gene Krox-20 is expressed in two alternating segments, rhombomeres (r) 3 and 5, in the developing mouse hindbrain. This expression pattern is established prior to rhombomere formation in the mouse, but it is not known how the timing of expression relates to cellular events of segmentation, such as lineage restriction. We have cloned Krox-20 sequences from Xenopus and the chick and shown that its alternating expression pattern is conserved in these systems, suggesting that its role in hindbrain development is conserved. Analysis of the early stages of Krox-20 expression in the chick show that both domains of expression precede the restriction of cell lineage to specific rhombomeres, consistent with a role of this gene in early events of hindbrain segmentation. The finding that expression is not coincident with lineage restriction indicates that early expression may not reflect an irreversible commitment of cells to r3 and r5 and/or may be mosaic.