Direct sequence analysis of proteins electroblotted from two-dimensional polyacrylamide gels onto immobilizing matrices provides an efficient technique for obtaining N-terminal sequence data for proteins not amenable to purification by reversed-phase high-performance liquid chromatography (RP-HPLC). We present in this paper a procedure for obtaining peptide fragments from electroblotted proteins for internal amino acid sequence analysis. First, Coomassie Blue-stained proteins are extracted from polydivinylidene difluoride membranes, using a detergent mixture of sodium dodecylsulfate and Triton X-100. Proteins are then separated from the detergent mixture by a chromatographic procedure which relies on the ability of proteins to interact with certain reversed-phase sorbents at high organic solvent concentrations. Under these conditions, detergents and Coomassie Blue are not retained and pass through the column. Proteins are recovered by simultaneously: (i) introducing trifluoroacetic acid into the mobile phase and (ii) decreasing the organic solvent concentration. After proteolytic fragmentation, peptides are purified by microbore-column (1-2 mm I.D.) RP-HPLC for microsequence analysis.