A slow reacting substance produced by rat basophilic leukemia cells, treated with ionophore A23187, was characterized by spectroscopic methods, enzymatic conversions, and chemical degradations as 5-hydroxy-6-S-cysteinylglycyl 7,9,11,14-eicosatetraenoic acid (leukotriene D). gamma-Glutamyltranspeptidase [gamma-glutamyltransferase; (5-glutamyl)-peptide: amino-acid 5-glutamyltransferase, EC 2.3.2.2] converted leukotriene C to a product identical to leukotriene D. This suggests that the stereochemistry of the arachidonyl moiety of leukotrienes C and D is the same [5(S)-6(R)-7,9-trans-11,14-cis]. Leukotriene D induces a faster contraction and, on a molar basis, is more potent than leukotriene C in the isolated guinea pig ileum bioassay.