Methods are described for isolating smooth muscle cells from the tracheae of adult and neonatal sheep and measuring the single-cell shortening velocity. Isolated cells were elongated, Ca²⁺ tolerant, and contracted rapidly and substantially when exposed to cholinergic agonists, KCl, serotonin, or caffeine. Adult cells were longer and wider than preterm cells. Mean cell length in 1.6 mM CaCl₂ was 194 ± 57 (SD) µm (n = 66) for adult cells and 93 ± 32 µm (n = 20) for preterm cells (P < 0.05). Mean cell width at the widest point of the adult cells was 8.2 ± 1.8 µm (n = 66) and 5.2 ± 1.5 µm (n = 20) for preterm cells (P < 0.05). Cells were loaded into a perfusion dish maintained at 35°C and exposed to agonists, and contractions were videotaped. Cell lengths were measured from 30 video frames and plotted as a function of time. Nonlinear fitting of cell length to an exponential model gave shortening velocities faster than most of those reported for airway smooth muscle tissues. For a sample of 10 adult and 10 preterm cells stimulated with 100 µM carbachol, mean (± SD) shortening velocity of the preterm cells was not different from that of the adult cells (0.64 ± 0.30 vs. 0.54 ± 0.27 s¹, respectively), but preterm cells shortened more than adult cells (68 ± 12 vs. 55 ± 11% of starting length, respectively; P < 0.05). The preparative and analytic methods described here are widely applicable to other smooth muscles and will allow contraction to be studied quantitatively at the single-cell level.