Respiration is modulated by lung mechanoreceptor feedback in vivo on a cycle-to-cycle basis. We replicated this modulation in vitro and tested four stimulus protocols to identify which of these most closely replicated in vivo responses to lung mechanoreceptor activation in mammals. We activated pulmonary vagal afferent pathways by electrical stimulation or by lung inflation, applied during expiration, which produces expiratory lengthening in vivo. In each modality, transient and tonic stimuli were applied. Stimuli were applied over a range of delays following inspiratory termination. Tonic stimuli were maintained until subsequent inspiratory onset. All stimulus modalities prolonged expiration (P < 0.05). These results indicate that the neural circuitry mediating pulmonary afferent modulation of expiratory duration is retained in vitro.