The expression of iron regulated proteins (IRPs) in vitro has been obtained in the past by adding iron chelators to the culture after bacterial growth, in the presence of an organic iron source. We have investigated aspects concerning full expression of the meningococcal IRPs during normal growth, in defined conditions using Catlin medium, Mueller Hinton and Tryptic Soy Broth (TSB). The expression of IRPs varied between different strains with respect to Ethylenediamine Di-ortho-Hidroxy-phenyl-acetic acid (EDDA) concentrations, and according to culture medium, and also between different lots of TSB. For each strain, a specific set of IRPs were expressed and higher EDDA concentrations, or addition of glucose, or use of different culture media did not resulted in a differential expression of IRPs. We were not able to grow N. meningitidis under normal growth conditions using Desferal. We looked for a good yield of outer membrane vesicles (OMVs) expressing IRPs in iron-deficient Catlin medium containing EDDA and Hemin. Culture for 32 h at 30 degrees C after growing for 16 h at 37 degrees C supported good bacterial growth. Bacterial lysis was noted after additional 24 h at 30 degrees C. Approximately 4 times more OMVs was recoverable from a culture supernatant after 24 h at 30 degrees C than from the cells after 16 h at 37 degrees C. The IRP were as well expressed in OMVs from culture supernatant obtained after 24 h at 30 degrees C as from the cells after 16 h at 37 degrees C.