Heparan sulfate (HS) proteoglycans modulate the biological activity of a number of growth factors in development, homeostasis, and cancer. Specific modifications of HS chains by HS biosynthetic enzymes have been implicated in growth factor signaling in multiple aspects of organogenesis. Although the role of HS 6-O-sulfotransferases has been described in processes such as trachea formation in Drosophila and vasculogenesis in zebrafish, little is known about how HS 6-O-sulfotransferases (Hs6st1-3 in mice) influence mouse development. To address this issue, we generated a conditionally mutant Hs6st1 mouse line and then generated mice with systemic inactivation of Hs6st1. Hs6st1-null pups were viable and grossly normal at birth. The lack of obvious abnormalities in lung, liver, and kidney, which express high levels of Hs6st1 during development, suggests that at least during embryonic life, the loss of Hs6st1 function may be compensated for by mechanisms involving other HS modifying enzymes. During early adulthood, however, Hs6st1-null mice failed to thrive and exhibited growth retardation, body weight loss, enlargement of airspaces in the lung and, in some cases, lethality. Our results suggest a potentially critical role for HS 6-O sulfation by Hs6st1 in postnatal processes. genesis 46:8–18, 2008. © 2008 Wiley-Liss, Inc.