Primary macrophages isolated from hck(-/-) igr(-/-) mice display altered morphology and F-actin cytoskeletal structures and reduced migration. The ability of phorbol myristyl acetate (PMA), a protein kinase C activator that has been reported to increase macrophage spreading and carcinoma cell motility, to rescue these hck(-/-) fgr(-/-) defects was tested. Although PMA-treated wild-type and hck(-/-) fgr(-/-) macrophages exhibited a similar flattened, spread phenotype, PMA did not rescue the hck(-/-) fgr(-/-) macrophage migration defect. Instead, both PMA-treated wild type and hck(-/-) fgr(-/-) macrophages were defective in spontaneous and chemotactic migration and tyrosine phosphorylation of the Cbl protooncoprotein was decreased in both. Moreover, c-cbl(-/-) macrophages displayed the same impairment of motility as hck(-/-) fgr(-/-) macrophages and a similar morphology with less polarization and more dorsal ruffling than wild-type macrophages. As Hck and Fgr expression and activity were not decreased in c-cbl(-/-) macrophages, these results suggest that Cbl is likely to be an important downstream mediator of the Src family kinase-regulated macrophage motility pathway. J. Cell. Physiol. 195: 276-289, 2003. (C) 2003 Wiley-Liss, Inc.