Upper airway muscles (UAM) are activated during inspiration, but little is known about their activity during expiration. We studied six healthy volunteers in supine position. Negative pressure (-10 to -30 cm H2O) was applied cyclically at the mouth during expiration. Subjects actively breathed during inspiration and either contracted or relaxed their UAM during expiration. Supraglottic pressure (Psg) was measured with a catheter with the tip at the supraglottic level. During relaxation, but not during contraction, there was expiratory flow limitation (FL) accompanied by high frequency pressure and flow oscillations (70 +/- 5.7 Hz at -30 cm H2O). The average linear supraglottic resistance was higher during relaxation than during contraction at both -10 and -20 cm H2O (p < 0.05), but not at -30 cm H2O. Amplitude of expiratory genioglossus electromyogram was 3.6 +/- 2.5 mm during quiet breathing, 7.0 +/- 3.8 mm during contraction (p < 0.05) and 4.1 +/- 1.8 mm during relaxation. Average mouth pressure, which immediately preceded pressure (and flow) oscillations was -2.1 +/- 0.9 cm H2O. In one subject cineradiography of the neck during expiratory application of -30 cm H2O showed anterior flexion and high frequency oscillations of the soft palate during relaxation. During contraction, the soft palate was close to the posterior pharyngeal wall without oscillations. We conclude that cyclically applied negative pressure at the mouth during expiration produced expiratory flow limitation during relaxation but not during contraction, and that expiratory contraction of UAM stabilizes upper airways during negative pressure application at the mouth.