Prostaglandins (PG) E2 and F2alpha, characterized by thin-layer chromatography and bioassay, were released from guinea-pig trachea contracted with acetylcholine or histamine. Inhibition of contraction with atropine or mepyramine, or by the removal of calcium, abolished the prostaglandin release. PGE2 and PGF2alpha were also released after gentle mechanical irritation of the mucosal surface, but not the adventitial surface, of the trachea. This release of prostaglandins occurred in the absence of calcium and was prevented by treatment of the trachea with indomethacin. Incubation (20 minutes) of tracheal spirals with indomethacin (0.6 mug/ml) 1) reduced the basal tension of the spiral; 2) reduced responses to low doses of histamine, serotonin, acetylcholine, barium or potassium; and 3) increased responses to high doses of these agonists. These effects lasted despite washout but were reversed by the addition of arachidonic acid. Subthresholdquantities of PGF2alpha after indomethacin treatment restored responses to minimally effective doses of the agonists. Aspirin (50 mug/ml), 5,8,11,14-eicosatetrayonic acid (2 mug/ml) and sodium salicylate (100 mug/ml) had effects similar to indomethacin (0.6 mug/ml). Alterations produced with 5,8,11,14-eicosatetrayonic acid and sodium salicylate were reversed with washing. Restoration of resting tension after indomethacin did not qualitatively change the results. Indomethacin at higher doses (greater than 30 mug/ml) inhibited responses to all histamine doses but this effect was reversible with washing. The results suggest that basal tension of the guinea-pig trachea may be due to an intramural production of PGF2alpha and that during the development of active tension, prostaglandins E2 and F2alpha are released which modulate the intensity of the contraction.