The peritoneal contact surface area (PCSA), which represents the area parameter in the mass transfer area coefficient (MTAC), is a crucial marker in the evaluation of peritoneal dialysis effectiveness. However, the capacity to recruit a larger PCSA has only been rarely demonstrated in vivo and, in most cases, changes in MTAC are interpreted as permeability changes and not as surface area variations. Here, we report the use of micro-computerized tomography (μCT) for the measurement of PCSA changes to various fill volumes. Using this three-dimensional imaging method, PCSA was measured in vivo in 26 healthy Wistar rats receiving intraperitoneally increasing fill volumes of peritoneal dialysis solutions: 5 mL (group 1, n = 8), 10 mL (group 2, n = 8) and 15 mL (group 3, n = 10) per 100 g of body weight. A non-ionic iodinated contrast agent was added to the dialysis solution in order to distinguish the intraperitoneal dialysis solutions from soft tissues. The normalized PCSA/weight ratio (cm2/g) increased with fill volume: 1.12 ± 0.10 cm2/g (range 0.98-1.25) in group 1; 1.74 ± 0.08 cm2/g (range 1.64-1.87) in group 2; 2.13 ± 0.09 cm2/g(range 1.90-2.30) in group 3. With this μCT method, PCSA recruited in vivo with a 10 mL/100 g fill volume was in the range 94-107%) of ex vivo total peritoneal surface area (evPSA), as calculated with the Kuzlan's formula. With a 15 mL/100 g fill volume, the in vivo-measured PCSA, the exchange surface area, surpassed the evPSA (range 113-139%).