Myeloid-differentiated HL-60 cells were used to study the activation of G-proteins by receptor agonists. Following incubation of membranes with the photoreactive GTP analog. [α- 3 2 P]GTP azidoanilide, and subsequent exposure to ultraviolet light (254 nm), photolabeling of 40 kDa proteins comigrating with the G i 2 α-subunit was observed. Photolabeling in the absence or presence of the chemoattractant, N-ionnyl-methionyl-leucyi-phenylalanin (FMLP), absolutely required Mg 2 + ; FMLP stimulated photolabeling at all Mg 2 + concentrations employed (up to 30 mM). Addition of GDP (3-50 μM) reduced basal photolabeling to a greater extent than photolabeling stimulated by FMLP. FMLP did not stimulate photolabeling of proteins modified by pertussis toxin. Leukotriene B 4 and C5a also stimulated photolabeling of 40 kDa proteins. The results indicate that (i) the major G-protein in HL-60 cells, G i 2 requires Mg 2 + for basal and receptor-stimulated activity, (ii) effective receptor-mediated activation of G-proteins is observed at mM concentrations of Mg 2 + , and (iii) receptor agonists apparently reduce the affinity of G-proteins for GDP.