Since the time-course of hyaluronan (HA) accumulation and fragmentation in inflammatory tissues remains unknown, our study was performed to analyse the expression of the main hyaluronidases and HA synthases, as well as HA fragmentation, after ischemia/reperfusion injury (IR) in the rat kidney. The expression of HYAL1, HYAL2, HAS1, HAS2 and HAS3 was evaluated by real-time PCR in the outer [OSOM] and inner stripes [ISOM] of the renal outer medulla up to Day 14 after IR injury. We also assessed HA fragmentation by membrane filtration followed by pseudo-ELISA method. HAS1 was up-regulated more than 50- and 35-fold in OSOM and ISOM respectively, at 12 h post-IR, returning to baseline faster in ISOM than in OSOM. HAS2 mRNA increased only after a 2-day delay and remained elevated until Day 14 post-IR (mRNA for HAS3 was not detected). Both HYAL1 and HYAL2 were strongly but transiently (12–48 h) repressed. The progressive HA accumulation in the post-ischemic kidney, detected by immunohistochemistry (Declèves et al., 2006), was confirmed by the peudo-ELISA method. Indeed, the amount of HA in control kidneys averaged 29+/­9 and 370+/­28 ng/mg in OSOM and ISOM, and was significantly enhanced to 1201+/­119 and 1524+/­132 ng/mg at Day 14 post-IR, respectively. However, the amount of low molecular weight HA (i.e., HA fragments) decreased by 40% at 24 h post-IR in ISOM. At later stages post-IR, the amount of HA fragments increased in both zones (x80 in OSOM and x6 in ISOM versus baseline, at Day 14). In summary, within 24 h post-IR, there is a massive activation of HAS1 combined with a repression of hyaluronidases, leading to a pool of predominantly high MW HA. Significant amounts of HA fragments appear later (Day 7–14), when HAS2 is induced and HYAL1 and HYAL2 levels are back to baseline. This modulation of HA breakdown after IR injury further suggest that this process might be involved in post-ischemic events such as inflammation and/or regeneration.