The objective of this investigation was to develop a method for real-time measurement of changes in luminal area in microexplants of airways during pharmacological and physiological interventions. After guinea pigs were killed, tracheal rings (1- to 2-mm thick) were excised and placed in 300-microliters chambers. The area of the airway lumen was calculated as pixel number with the use of computerized videomicrometry. In 29 epithelium-intact airways, 10(-3) M acetylcholine (ACh) caused decrease in luminal area of 38.1 +/- 2.80% (P < 0.001 vs. 10(-9) M). Spontaneous tone also was demonstrated in 34 preparations from 4 guinea pigs; decrease in area of 17.0 +/- 1.45% after 60-min incubation in buffer alone was blocked completely by 10(-5) M indomethacin (P = 0.01). Luminal narrowing caused by < or = 10(-6) M ACh was reversed completely by 10(-6) M albuterol (P = 0.002). Addition of 100,000 activated human eosinophils caused 24.7 +/- 4.41% decrease in luminal area vs. 7.24 +/- 5.51% for nonactivated cells (P = 0.048). We demonstrate a real-time method for the assessment of auxotonic changes in airway caliber that utilizes microsections of explanted airways and permits the use of extremely small numbers of isolated cells to achieve physiological activation. Concentration-response characteristics and spontaneous tone are similar to those of large chamber preparations, and narrowing is reversed by beta 2-adrenoceptor activation.