The importance of the interstitium and its major ground substance component, hyaluronan (HA), for solute and fluid transport across the peritoneal membrane has been debated during the last few years. We therefore partly removed HA from the peritoneal membrane using enzymatic digestion with hyaluronidase for 2 h, after which the transport properties of the peritoneal membrane were studied in peritoneal dialysis dwells. A dialysis fluid containing 3.86% glucose was used. As a marker of macromolecular transport, the total peritoneal clearance of radiolabelled albumin out of the peritoneal cavity and its clearance to plasma were measured, as well as the albumin clearance from plasma to dialysate. Transport of small solutes between plasma and dialysate was measured by assessing the mass transfer area coefficient of 51Cr-EDTA and glucose. Hyaluronidase preincubation yielded a 78% reduction of HA in the superficial layer of the peritoneal membrane, without alterations in the transport of either small or large solutes compared with the situation in preincubated controls. The only changes observed were between rats incubated with either hyaluronidase or vehicle alone compared to non-incubated controls. In conclusion, despite a large reduction of the HA content of the tissues surrounding the peritoneal cavity, hyaluronidase incubation did not produce any significant changes in solute and fluid transport across the peritoneal membrane. Our data indicate that peritoneal membrane HA in physiological concentrations plays a rather subordinate role in the overall transport of small solutes and water across the capillary–interstitial peritoneal barrier.