Prostaglandin D2 (PGD2) concentration-dependently induced a contraction of the longitudinal muscle of the intact isolated rat colon in vitro. The effect of PGD2 increased continuously from the anal to the oral end of the colon. The action of PGD2 was not inhibited but rather enhanced by the neurotoxin, tetrodotoxin, and by the PGD2 antagonist, AH 6809. In contrast, the thromboxane A2 antagonist, SK&F 88046, concentration-dependently inhibited the PGD2 effect. The action of PGD2 was mimicked by the stable thromboxane A2 derivative, carbocyclic thromboxane A2, indicating that PGD2 exerts its action on the smooth muscle by stimulation of thromboxane receptors. Between 18 (distal colon) and 38% (proximal colon) of the preparations exhibited spontaneous phasic myogenic contractions. The thromboxane antagonist, SK&F 88046, completely suppressed these spontaneous contractions. The combined lipoxygenase/cyclo-oxygenase inhibitor nordihydroguaiaretic acid and sulfasalazine mimicked the action of SK&F 88046, whereas the cyclo-oxygenase inhibitor, indomethacin, was ineffective. These results suggest that endogenously produced metabolites of arachidonic acid, e.g. thromboxane A2, contribute to the generation of spontaneous muscle contractions in vitro. The failure of indomethacin to suppress muscular activity, however, requires further studies.