Hypertrophy and hyperplasia of airway smooth muscle (ASM) cells are features of asthma that can be assessed in airway transverse sections using stereologically derived parameters. However, little is known about the variability of these parameters within and between airways. The aim of the present study was to assess sources of variation in the measurement of the area of the ASM layer (AASM), and the volume fraction of ASM cells (VVASM) and numerical density of ASM cells within that layer. AASM increased by up to 12% in 4-μm sections, and 28% in 30-μm sections, compared with 0.5-μm sections. AASM was greater (P < 0.05) in large than in small airways, and varied by up to 28% along segments of large airways. Numerical density of ASM cell estimates around the airway circumference varied by less than 10% if 40 random high-power fields were sampled. VVASM was most accurately estimated on 0.5-μm, rather than 4- or 30-μm sections, and was less (P < 0.05) in large than in small airways. The coefficients of variation for VVASM were less than 10% along airway segments. We found that variation of parameters used to estimate ASM cell number or size could be minimized with adequate sampling frequency around or along airway segments. Section thickness was positively related to the measured area of ASM on transverse airway sections. Thin (0.5-μm) sections should be used to estimate tissue volume fractions, which vary little within and between airways of similar size. Airway size contributes most to the variation in estimating parameters of the ASM layer.