We used multiple-breath Na washouts (MBNW) to study the inhomogeneity of ventilation in four normal humans (mean age 42.5 yr) before, during, and after 9 days of exposure to microgravity on Spacelab Life Sciences-1. Subjects performed 20-breath MBNW at tidal volumes of similar to 700 ml and 12-breath MBNW at tidal volumes of similar to 1,250 ml. Six indexes of ventilatory inhomogeneity were derived from data from 1) distribution of specific ventilation (SV) from mixed-expired and 2) end-tidal N-2, 3) change of slope of N-2 washout (semilog plot) with time, 4) change of slope of normalized phase III of successive breaths, 5) anatomic dead space, and 6) Bohr dead space. Significant ventilatory inhomogeneity was seen in the standing position at normal gravity (1 G). When we compared standing 1 G with microgravity, the distributions of SV became slightly narrower, but the difference was not significant. Also, there were no significant changes in the change of slope of the Na washout, change of normalized phase III slopes, or the anatomic and Bohr dead spaces. By contrast, transition from the standing to supine position in 1 G resulted in significantly broader distributions of SV (P < 0.05) and significantly greater changes in the changes in slope of the N-2 washouts (P < 0.001), indicating more ventilatory inhomogeneity in that posture. Thus these techniques can detect relatively small changes in ventilatory inhomogeneity. We conclude that the primary determinants of ventilatory inhomogeneity during tidal breathing in the upright posture are not gravitational in origin.