The transport of macromolecules through the lung interstitium depends on both bulk transport of fluid and diffusion. In the present study, we studied the diffusion of albumin. Isolated rabbit lungs were inflated with silicon rubber via airways and blood vessels, and two chambers were bonded to the sides of a 0.5-cm-thick slab that enclosed a vessel with an intersititial cuff. One chamber was filled with either albumin solution (2 or 5 g/dl) containing tracer ¹²⁵I-albumin or with tracer ¹²⁵I-albumin alone; the other was filled with Ringer solution. Unbound ¹²⁵I was removed from the tracer by dialysis before use. The chamber with Ringer solution was placed in the well of a NaI(Tl) scintillation detector. Diffusion of tracer through the interstitium was measured continuously for 60 h. Tracer mass (M) showed a time (t) delay followed by an increase to a steady-state flow (dM/dt constant). Albumin diffusion coefficient (D) was given by L²/(6T), where T was the time intercept of the steady-state M-t line at zero M, and L was interstitial length. Interstitial cuff thickness-to-vessel radius ratio (Th₀/R) was estimated by using Fick's law for steady-state diffusion. Both D and Th₀/R were independent of albumin concentration. D averaged 6.6 × 10⁷ cm²/s, similar to the free D for albumin. Values of Th₀/R averaged 0.047 ± 0.024 (SD), near the values measured histologically. Thus pulmonary interstitial constituents offered no restriction to the diffusion of albumin.