Two methods have been used to test for the presence of extravascular protein in the interstitium of the renal inner medulla. First, ascending vasa recta (AVR) segments were perfused with buffer containing 5 g/dl of albumin. The hydraulic pressure in the perfused vessel was varied to control transmembrane volume flux (Jv) to the interstitium. Interpolation to the point of zero Jv was employed to estimate interstitial Starling forces in the hydropenic rat papilla. Analysis of those experiments predicts that interstitial protein concentration (Ci) is high. When AVR segments are filled with oil, the oil column spontaneously breaks up as fluid is secreted into the lumen from the papillary interstitium. To obtain a lower limit on Ci, isolated AVR segments (IAS) filled with oil were sampled to measure protein concentration in the secreted fluid. In hydropenic rats, protein concentration was 3.4 +/- 0.5 and 5.2 +/- 0.2 g/dl in IAS and adjacent free-flowing AVR, respectively (P < 0.01). In rats subjected to furosemide and saline diuresis, the values were nearly identical, 4.7 +/- 0.2 and 5.2 +/- 0.2 g/dl, respectively. These separate experimental approaches corroborate a high concentration of protein in the renal inner medullary interstitium.