Platelet-activating factor (PAF-acether), a known platelet stimulant and bronchoconstrictor (in vivo), is a potential mediator of inflammation and thrombosis. However, all smooth muscle effects of PAF-acether described to date are indirect, relying upon intravascular platelet activation. Novel actions of PAF-acether and serotonin (5-HT) are presented here; these actions may lead to the development of a practical bioassay for PAF-acether and contribute to the understanding of the mechanism of action for both substances. PAF-acether, when added to a spiral cut guinea-pig trachea suspended in a tissue bath containing Krebs-Henseleit buffer, produced a dose-dependent loss of active tissue tension. The ED50 for this effect of PAF-acether was 75 ng/ml. PAF-acether produced a maximal relaxation which was 68% of that produced by PGE1 and the effect could not be modified by aspirin or propranolol pretreatment. 5-HT, alone, contracted the guinea-pig trachea strip in a dose-dependent manner, but caused relaxation instead when methysergide was present. Aspirin, phenoxybenzamine and propranolol did not alter this loss of active tissue tension. A similar observation was made in vivo using the guinea-pig bronchoconstriction model, in which PAF-acether as well as 5-HT given to methysergide-treated animals caused a decrease in intratracheal pressure. This action of PAF-acether may yield a suitable bioassay method which could facilitate routine measurements of the substance. Furthermore, the similarity in action of PAF-acether and of 5-HT on methysergide-treated animals leads one to speculate about the relationship between the two substances and their mechanism of action in smooth muscle.