An improved and simplified procedure for enzymatic digestion of proteins bound to polyvinylidene difluoride (PVDF) membranes for obtaining internal protein sequence data is presented. This improved procedure is compatible with various enzymes (trypsin, endoproteinase Lys-C, endoproteinase Glu-C, and clostripain) and is performed in the presence of 1% hydrogenated Triton X-100 (RTX-100)/10% acetonitrile/100 mM Tris, pH 8.0, followed by microbore HPLC purification of the recovered peptides. Previously published techniques required treatment of the PVDF-bound protein with polyvinylpyrrolidine M(r) 40,000 (PVP-40) prior to digestion, in order to prevent adsorption of the enzyme to the membrane. Unfortunately, contaminants produced from residual PVP-40 interfere with subsequent peptide mapping. We have found that when RTX-100 is used in the digestion buffer, no pretreatment of the PVDF-bound protein with PVP-40 is necessary. Advantages of this improved (one-step) procedure over the two-step PVP-40 procedure are (a) the elimination of undesirable contaminants associated with PVP-40, (b) a decrease in the time required for the technique, and (c) a reduction in sample manipulation. Peptide maps and recoveries from PVDF-bound standard proteins (4 micrograms each) enzymatically digested with this one-step method are compared with those obtained from the standard PVP-40 method. In addition, peptide maps and internal sequence data from low-level quantities of unknown proteins enzymatically digested with the improved procedure are presented. To date, this improved, one-step procedure has been successfully applied to 52 PVDF-bound unknown proteins (0.7-10 micrograms) of varying molecular weight (19-300 kDa) for which internal sequence data were obtained.