Hyaluronan is a large glycosaminoglycan that is abundant in the interstitium of the renal medulla/papilla. Papillary hyaluronan increases during hydration and decreases during dehydration. Due to its gel properties and ability to retain large volumes of water, hyaluronan plays a role in renal water handling by affecting the permeability characteristics of the papillary interstitium. The focus of the present investigation was the regulation of hyaluronan metabolism in the kidney, especially during variations in hydration status. In control papillas, HAS 2 mRNA was heavily expressed and HAS 1 and 3 mRNA were weakly distributed. HYALs 1–3 mRNA were found at high expression and HYAL 4 was only weakly expressed. In hydrated animals, the diuretic response (12-fold) was followed by a 58% elevation in papillary hyaluronan and a 45% reduction in the excreted urinary hyaluronidase activity. No difference was determined in HAS 1–3 mRNA or HYAL 1, 3–4 mRNA expression, suggesting a change in activity rather than amount of protein. In dehydrated animals, antidiuresis was followed by a 22% reduction in papillary hyaluronan and a 62% elevation in excreted urinary hyaluronidase activity. Plasma vasopressin was 2.8-fold higher in dehydrated vs. hydrated rats. In conclusion, HAS 2 appears a major contributor to the baseline levels of hyaluronan. Reduced HAS 2 gene expression and increased excreted urinary hyaluronidase activity during dehydration contribute to the reduced amount of hyaluronan and to antidiuretic response.