We identified a 39 kilodalton (kDa) protein from newly molted American cockroaches (Periplaneta americana), isolated from a 100,000 g precipitate of a 1000 g supernatant from an integument homogenate, which exhibited increased phosphorylation following diflubenzuron (DFB) treatment. This 39 kDa phosphoprotein was partially purified from an intracellular membrane vesicle containing fractions obtained by discontinuous sucrose density centrifugation. Both the interfaces of the 30/40% and 40/50% sucrose density layers were found to be enriched in the 39 kDa phosphoprotein. Treatments with various chemicals such as the ionophores valinomycin and A23187, the protonophore carbonylcyanide p-trifluoromethoxy-phenylhydrazone (FCCP), and the vacuolar H(+)-ATPase inhibitor N-ethylmaleimide (NEM), like DFB, were also found to stimulate phosphorylation of 39 kDa protein. These results suggest that by disrupting the normal pH gradient occurring in certain acidic vacuolar-type membrane vesicles, phosphorylation of the 39 kDa protein will be increased. In addition, we found that by decreasing the external pH to 5.0 from about neutral, it was possible to stimulate the phosphorylation activity of this particular protein, and thus stimulate the action of DFB. We concluded the DFB's action is very probably related to its ability to disrupt the normal ionic balance of these vacuolar-type vesicles, leading to eventual disruption of the proton gradient within the vesicles.