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It is currently proposed that accumulation of hyaluronic acid (HA) and subsequent hydration of the cardiac extracellular matrix is required for normal looping of the vertebrate heart. To test this hypothesis, we cultured Wistar rat embryos (Gestational Day 9.5) in rat serum plus 20 TRU/ml of Streptomyces hyaluronidase (treated embryos) or rat serum alone (control embryos). Despite degradation of HA as documented by Alcian blue staining at pH 2.5, 57 of 59 treated embryos developed normally looped hearts after 36 hr in culture. These experiments suggest that the accumulation of HA is not required for normal looping of the mammalian heart in situ.