Background: Hox genes belong to a highly conserved subgroup of the homeobox gene superfamily. Studies of animal models have emphasized their role in defining the body plan by their coordinated expression along the body axis during ontogeny. Although an important role of HOX genes in human development is assumed, little is known about their expression during human ontogenesis. Therefore, we investigated the expression of the nine most posterior members of the HOXA, HOXB, HOXC, and HOXD clusters in embryonic hindgut between weeks 6 to 12 and in adult rectal tissue. Results: Applying in situ hybridization and immunohistochemistry, we observed expression of HOXA11, HOXA13, HOXD12, and HOXD13 in developmental week 6. However, expression of HOXD12 faded during weeks 7 and 8, and then became increasingly re-expressed during week 9 in humans. With the exception of HOXD13, all expressed HOX genes dropped below detection limits in week 11. Adult rectal tissue displayed distinct HOXA11, HOXA13, HOXD12, and HOXD13 expression patterns within the rectal layers. Conclusions: Our data suggest a strict spatio-temporal regulation of HOX gene expression during human development, supporting the idea of their role as key regulators. Nonetheless, the expression pattern of distinct HOX genes differs markedly from animal models. Developmental Dynamics 242:53–66, 2013. © 2012 Wiley Periodicals, Inc.