Stimulation of monoblastic U937 cells with transforming growth factor β1 and 1,25‐(OH)2 vitamin D3 (TGF‐β1/D3) upregulates urokinase receptor (uPAR) and confers urokinase‐dependent adhesiveness to the cells for serum‐or vitronectin‐coated surfaces. Recent studies show that uPAR itself is a high‐affinity adhesion receptor for vitronectin and that urokinase (uPA) is an activator of this adhesive function. In the course of exploring possible G‐protein involvement in this adhesion it was observed that TGF‐β1/D3‐primed U937 cells became adhesive to vitronectin in an uPAR‐dependent manner when exposed to pertussis toxin (PTX). The adherent response is concentration‐ and time‐dependent, and was not due to the ADP‐ribosyltransferase activity of the toxin because the purified B‐subunit of PTX was equally effective. Although promoting adhesion to serum‐ or vitronectin‐coated surfaces, PTX blocked spontaneous cell adhesion to fibrinogen, an endogenous ligand for the Mac‐1 receptor (CD11b/CD18). Flow cytometry study showed that expression of the α‐subunit of Mac‐1 (CD11b) on primed cells was increased by nearly threefold. Monoclonal antibody to CD11b abolished the PTX‐induced cell adhesion and the binding of the primed cells to PTX‐coated plates. Activation of Mac‐1 receptor by its endogenous ligand fibrinogen induced cell adherent response similar to PTX. PTX, but not uPA, triggered a rapid rise in [Ca2+]i in primed U937 cells, and PTX‐induced adhesion was significantly attenuated by 1,2‐bis‐(2‐aminophenoxy)ethane‐NNN ′,N ′‐tetraacetic acid/acetoxy‐methyl ester (BAPTA/AM), a selective membrane‐permeant [Ca2+]i chelator. PTX‐induced cell adhesion was also prevented by antibodies to uPAR and by conditioned medium containing soluble uPAR. Together these data indicate that PTX B‐subunit may bind to Mac‐1 integrin, which leads to a rapid rise in [Ca2+]i and subsequent activation of uPAR for adherence to vitronectin, suggesting a functional link between Mac‐1 and activation of uPAR important to cellular trafficking and host defence in response to Bordetella pertussis infection.