The solubilization of a particular protein is mandatory for its subsequent resolution and detection in two-dimensional gels. However, the extraction solutions, that are compatible with the first-dimensional separation step, such as urea and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), do not solubilize all proteins in a sample. We studied the effect of various common, strong detergents and chaotropes, widely used as solubilizing agents, such as sodium dodecyl sulfate, lithium dodecyl sulfate and guanidine hydrochloride, on the solubilization of the total and membrane proteins of the bacterium Haemophilus influenzae. The proteins solubilized with each system were analyzed by two-dimensional electrophoresis and these of interest were identified by matrix assisted laser desorption/ionization-mass spectrometry (MALDIMS). Use of sodium dodecyl sulfate, lithium dodecyl sulfate or guanidine hydrochloride for the solubilization of total proteins of the microorganism resulted in the detection of several additional spots, representing mainly outer membrane proteins, in comparison with those detected in the soluble protein fraction. Solubilization of the proteins of the cell envelope fraction with sodium dodecyl sulfate did not result in a more efficient protein detection when compared to the extraction with the urea/CHAPS system. When the dry immobilized pH gradient strips were rehydrated in a solution containing the proteins of the membrane fraction solubilized with sodium dodecyl sulfate or lithium dodecyl sulfate, a larger number of protein spots were detected in comparison with strips that were rehydrated in the urea/CHAPS solution. However, no improvement was observed in comparison with protein application in sample cups. The additional proteins detected with the use of strong detergents and chaotropes are in the majority difficult to solubilize and less hydrophobic proteins.