We report here the effect of hydroxyurea (HU), which is an inhibitor of DNA synsthesis and a known cytotoxic and teratogenic agent, on the composition of glycosaminoglycans (GAGs) in the extracellular matrix of the developing forelimb of the rat. HU injected on day 12 has been shown to produce one missing digit and multiple missing digits on day 13 (Ritter et al., '73). Pregnant Wistar rats were treated (IP) with 1,000 mg/kg HU on either day 12 or day 13 (9:00 a.m.) of gestation and sacrificed 0–96 hours later. Forelimbs were dissected and pooled by litter. GAGs were extracted and then separated by microzone electrophoresis on cellulose acetate membranes. Identification of GAG was confirmed by digestion with specific polysaccharidases. Such analysis of control forelimb-buds revealed hyaluronic acid decreasing from 62% of total GAG at day 12 to 36% total GAG at day 16, whereas chondroitin sulfate increased from 34% to 58%. HA content of forelimb-buds treated on day 12 was 22% greater than controls at 12 hours posttreatment, 45.5% at 24 hours posttreatment, returning to control levels at 60 hours after treatment. Chondroitin sulfate (CS) level decreased by 20% at 48 hours after treatment, returning to control level by 72 hours. Hyaluronate levels of HU 13 treated forelimb-buds were 46% greater than controls at 24 hours after HU treatment, returning to control levels at 36 hours. CS levels were significantly lower than controls at 36 hours posttreatment. Histochemical analysis was performed to localize these alterations in GAG composition within the forelimbs. Beginning 12 hours posttreatment and continuing until 48 hours, an increase in HA content was observed in the mesenchymal compartment of the forelimb (1) beneath the apical ectodermal ridge and (2) between the ectoderm and prechondrogenic blastema (peripheral zone). This HA increase may be interpreted as a recovery (repair) phase of the pathogenesis of the HU malformation. To confirm that this shift in GAG is indicative of a compensatory phenomenon mitotic indices of the forelimbs were examined. At 36 hours posttreatment substantial increase in mitotic activity was observed in the subridge and peripheral regions of forelimbs treated on day 12, whereas those treated on day 13 demonstrated a significant increase only in the subridge zone. The increase in HA composition in the zones of the forelimb correlated well with the increase of mitotic activity. It is suggested that elevated HA/CS ratio allowed for increased proliferation and reorganization of the mesenchymal cell population, whereas the return to control levels marked the cessation of repair and resumption of differentiation.