Tissue factor (TF) procoagulant activity (PCA) on the surface of intact HL-60 cells is encrypted. This latent TF PCA was activated bv exposing the cells to ionomycin, a calcium ionophore. Within seconds an increase in TF PCA of greater than 100-fold was observed. The ionomycin effect was blocked by pretreating the cells with calmidazolium, a calmodulin inhibitor. Changes in TF structure and function, coincident with the ionophore-induced increase in TF PCA, were identified. TF-factor VIIa complexes formed on both untreated and ionophore-treated cells, but pseudosubstrate inhibitors only bound to TF- factor VIIa on the ionophore-treated cells. TF PCA was inhibited by reacting cells with sulfosuccinimidyl-6-(biotinamido)haxanoate, and the rate of this reaction increased twofold after cells were exposed to ionomycin. When proteins on the surface of untreated cells, expressing minimal TF PCA, were cross-linked with 3-3'-dithiobis(sulfosuccinimidylpropionate), cross-linked TF dimers were produced. TF cross-linking was prevented by first treating the cells with ionomycin. These results suggest a mechanism for the ionomycin- induced increase in TF PCA. TF activation appears to be e calmodulin- dependent process, which exposes an essential macromolecular substrate binding site on TF, possibly as the result of a change in TF quaternary structure.