Current techniques to estimate nitric oxide (NO) production and elimination in the lungs are inherently nonspecific or are cumbersome to perform (multiple-breathing maneuvers). We present a new technique capable of estimating key flow-independent parameters characteristic of NO exchange in the lungs: 1) the steady-state alveolar concentration (Calv,ss), 2) the maximum flux of NO from the airways ( J NO,max), and 3) the diffusing capacity of NO in the airways ( D NO,air). Importantly, the parameters were estimated from a single experimental single-exhalation maneuver that consisted of a preexpiratory breath hold, followed by an exhalation in which the flow rate progressively decreased. The mean values for J NO,max, D NO,air, and Calv,ss do not depend on breath-hold time and range from 280–600 pl/s, 3.7–7.1 pl · s−1 · parts per billion (ppb)−1, and 0.73–2.2 ppb, respectively, in two healthy human subjects. A priori estimates of the parameter confidence intervals demonstrate that a breath hold no longer than 20 s may be adequate and that J NO,max can be estimated with the smallest uncertainty and D NO,air with the largest, which is consistent with theoretical predictions. We conclude that our new technique can be used to characterize flow-independent NO exchange parameters from a single experimental single-exhalation breathing maneuver.