Direct analysis of proteins adsorbed onto the surface of nylon membranes has been performed at the picomole level by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Nylon-66 and positive charge-modified nylon (Zetabind) membranes fixed to MALDI probe tips were successfully employed to analyze picomole quantities of sample that were adsorbed onto these inert supports prior to adding a matrix-containing solution. Proteins and peptides are readily solubilized from these types of membrane with conventional matrix solvents and cocrystallize with the matrix on the membrane surface. Because solubilization of membrane-adsorbed protein is necessary for successful sample preparation, nylon membranes are more suitable for use with MALDI-MS than other protein transfer membranes such as polyvinylidene difluoride or nitrocellulose. When compared to samples prepared conventionally, no apparent loss of sensitivity or resolution is observed when analysis by MALDI-MS is performed from nylon-66 or positive charge-modified nylon membranes. Detection limits and resolution are not apparently affected by the membrane immobilization/washing procedure, and no change in the mass accuracy is observed when analysis is performed on the nylon surface. However, there is a time shift (increase) in ion flight time when analysis by MALDI-time-of-flight-MS is performed directly from the membrane fixed to the probe tip (about 200 ns for an ion of mass 379.3). To maintain mass accuracy, the use of internal standards or external calibration performed on a membrane support was necessary. The immobilization of proteins on nylon membranes can be used to facilitate removal of water-soluble contaminants because the sample is retained when the membrane is immersed in water prior to adding the matrix solution. The feasibility of performing both chemical and enzymatic modifications of proteins adsorbed onto inert nylon supports prior to analysis by MALDi-MS is also demonstrated.