We describe an optical method to quantify single- gland secretion. Isolated tracheal mucosa were mounted at the air-Krebs interface and coated with oil. Gland secretions formed spherical bubbles that were digitally imaged at intervals, allowing rates of secretion to be calculated. We monitored 340 glands in 54 experiments with 12 sheep. Glands secreted basally at low rates (0.57 ± 0.04 nl · min⁻¹ · gland⁻¹, 123 glands) in tissues up to 9 h postharvest and at lower rates for up to 3 days. Carbachol (10 μM) stimulated secretion with an early transient and a sustained or oscillating phase. Peak secretion was 15.7 ± 1.2 nl · min⁻¹ · gland⁻¹ (60 glands); sustained secretion was 4.5 ± 0.5 nl · min⁻¹ · gland⁻¹ (10 glands). Isoproterenol and phenylephrine (10 μM each) stimulated only small, transient responses. We confirmed that cats have a large secretory response to phenylephrine (11.6 ± 3.7 nl · min⁻¹ · gland⁻¹, 12 glands), but pigs, sheep, and humans all have small responses (<2 nl · min⁻¹ · gland⁻¹). Carbachol-stimulated peak secretion was inhibited 56% by bumetanide, 67% by HCO replacement with HEPES, and 92% by both. The distribution of secretion rates was nonnormal, suggesting the existence of subpopulations of glands.