The perimeter of the basement membrane (Pbm) of an airway viewed in cross-section is used as a marker of airway size since in normal lungs it is relatively constant, despite variations in airway smooth muscle (ASM) shortening and airway collapse. In vitro studies have suggested that differential stretch of the Pbm between asthmatic and non-asthmatic airways fixed in inflation may occur and lead to an over-estimation of ASM thickness in asthma. The relationships between the Pbm and the area of ASM were compared in transverse sections of airways from cases of fatal asthma (F) and from non-asthmatic control (C) where the lung tissue had been fixed inflated (Fi; Ci) or un-inflated (Fu; Cu). When all available airways were used, the regression slopes were increased in Fu and Cu, compared with Fi and Ci, and increased in Fu and Fi, compared with Cu and Ci, suggesting effects of both inflation and asthma group, respectively. When analyses were limited to airway sizes that were available for all groups (Pbm < 15 mm), the slopes of Fi and Fu were similar but both were greater than Ci and Cu, which were also similar. It was calculated that the effect of asthma group accounted for 80% and inflation for 20% of the differences between Fi and Ci. We conclude that the effects of inflation on the relationship between Pbm and ASM are small and do not account for the differences observed in ASM between cases of asthma and non-asthmatic controls.