We investigated the differential effect of histamine and methacholine on spirometry and ventilation distribution (where indexes S _cond and S _acin represent conductive and acinar ventilation heterogeneity; Verbanck S, Schuermans D, Van Muylem A, Noppen M, Paiva M, and Vincken W. J Appl Physiol 83: 1807–1816, 1997). Thirty normal subjects were challenged with cumulative doses of 6.52 μmol histamine and, on a separate day, with either 6.67 μmol methacholine (equal-dose group; n = 15) or 13.3 μmol methacholine (double-dose group; n = 15). Largest average forced expiratory volume in 1 s (FEV₁) decreases or S _cond increases obtained in either group were −9% and +286%, respectively; S _acin remained unaffected at all times. In the equal-dose group, a smaller FEV₁ decline ( P= 0.002) after methacholine was paralleled by a smaller S _cond increase ( P = 0.041) than with histamine. However, in the double-dose group, methacholine maintained a smaller FEV₁ decline ( P = 0.009) while inducing a larger S _cond increase ( P = 0.006) than did histamine. The differential action of histamine and methacholine is confined to the conductive airways, where histamine likely causes the greatest overall airway narrowing and methacholine induces the largest parallel heterogeneity in airway narrowing, probably at the level of the large and small conductive airways, respectively. The observed ventilation heterogeneities predict a risk for dissociation between ventilation-perfusion mismatch and spirometry, particularly after methacholine challenge.