The effects of benzylaminopurine (BAP) on the polypeptide pattern of membranes fromCatharanthus roseuscells were investigated using two- dimensional gel electrophoresis. In order to characterize the membrane preparation microsomes, issued from cells grown in different hormonal conditions (+2,4-D, -2,4-D, BAP), microsomes were harvested at different times of cell culture and tested for their ATPases prior to 2-D PAGE analysis. Inhibitory studies revealed that the total ATPase activity within these membranes was slightly affected by sodium azide and nitrate (less than 5%) indicating that mitochondrial and tonoplat vesicles were little represented in the crude fractions. Membranes contained, however, ATPases that were totally dependent on magnesium and strongly inhibited by orthovanadate (90% inhibition). Analysis by 2-D PAGE of the protein composition of these membranes revealed that the overall pattern was conserved after cytokinin treatment. A few polypeptides appeared, however, to be regulated by BAP. Particularly, the accumulation of a polypeptide with an apparent molecular mass of 33 kDa (P33) was increased markedly after cytokinin treatment. This stimulation was slightly inhibited by 2,4-D, a known inhibitor of alkaloid accumulation. Time course studies indicated that the effect of cytokinin on the accumulation of polypeptide P33 starts within 24 hours of cytokinin treatment and persists for at least 96 hours.