Activation of calpains by calcium flux leading to talin cleavage is thought to be an important process of LFA-I activation by inside-out signalling. Here, we tested the effects of the calcium ionophore ionomycin and calpain inhibitor calpeptin on LFA-1-mediated adhesion of a T cell hybridoma line, cytotoxic T cells and primary resting T cells. lonomycin activated LFA-1-mediated adhesion of all three types of T cells, and calpeptin inhibited the effects of ionomycin. However, calpeptin also inhibited activation of LFA-1 by PMA, which did not induce calcium flux. Cleavage of talin was undetectable inonomycin-treated T cells. Furthermore, treatment with ionomycin and calpeptin induced apoptosis of T cells. Inhibitors of phosphatidyl Inositol-3 kinase inhibited activation of LFA-I by ionomycin, but not by PMA, whereas the protein kinase C inhibitor inhibited the effects of PMA, but not ionomycin. Thus, activation of LFA-I by ionomycin is independent of calpain-mediated talin cleavage. (c) 2007 Elsevier Inc. All rights reserved.