The spatial and temporal co-linear expression of Hox genes during development is an exquisite example of programmed gene expression. The precise mechanisms underpinning this are not known. Analysis of Hoxb chromatin structure and nuclear organisation, during the differentiation of murine ES cells, has lent support to the idea that there is a progressive 'opening' of chromatin structure propagated through Hox clusters from 3'to 5', which contributes to the sequential activation of gene expression. Here, we show that similar events occur in vivo in at least two stages of development. The first changes in chromatin structure and nuclear organisation were detected during gastrulation in the Hoxb1-expressing posterior primitive streak region: Hoxb chromatin was decondensed and the Hoxb1 locus looped out from its chromosome territory, in contrast to non-expressing Hoxb9, which remained within the chromosome territory. At E9.5, when differential Hox expression along the anteroposterior axis is being established, we found concomitant changes in the organisation of Hoxb. Hoxb organisation differed between regions of the neural tube that had never expressed Hoxb [rhombomeres (r) 1 and 2], strongly expressed Hoxb1 but not b9 (r4), had downregulated Hoxb1 (r5), expressed Hoxb9 but not Hoxb1 (spinal cord), and expressed both genes (tail bud). We conclude that Hoxb chromatin decondensation and nuclear re-organisation is regulated in different parts of the developing embryo, and at different developmental stages. The differential nuclear organisation of Hoxb along the anteroposterior axis of the developing neural tube is coherent with co-linear Hox gene expression. In early development nuclear re-organisation is coupled to Hoxb expression, but does not anticipate it.