To test the hypothesis that during the course of a multiple-breath N2 washout (MBNW) diffusion-dependent ventilation maldistribution is more apparent in the early breaths, whereas convection-dependent maldistribution predominates in the later breaths, we performed MBNW with 0-, 1-, and 4-s end-inspiratory breath holds (BH0, BH1, BH4, respectively) in five normal subjects. Each subject breathed with a constant tidal volume of 1 liter, at 10-12 breaths/min and at constant flow rates. For each breath we computed the slope of the alveolar plateau normalized by the mean expired N2 concentration (Sn), the Bohr dead space (VDB), and an index analogous to the Fowler dead space (V50). In all five subjects, Sn, VDB, and V50 decreased with breath holding, indicating diffusion dependence of these indexes. Over the first five breaths the rate of increase of Sn as a function of cumulative expired volume (delta Sn/delta sigma VE) decreased by 29 and 54% during BH1 and BH4, respectively, compared with BH0. In contrast, from breath 5 to the end of the washout there was no significant change in delta Sn/delta sigma VE during BH1 and BH4 compared with BH0. Our results provide further experimental support for the hypothesis that the increase of Sn as a function of cumulative expired volume after the fifth breath constitutes a diffusion-independent index of ventilation inhomogeneity. It therefore reflects alveolar gas inequalities among larger units.(ABSTRACT TRUNCATED AT 250 WORDS)