This paper describes use of a novel glass bead-based immobilized-enzyme micro column for simple and swift on-line protein digestion then peptide separation by reversed-phase HPLC. The inexpensive and easily made immobilized-enzyme micro column was prepared from aminopropyl controlled-pore glass that was reacted first with glutaraldehyde then with trypsin in the presence of phosphate buffer. Tryptic digestion of bovine serum albumin (BSA) was achieved simply by passing pretreated protein solution through the laboratory made immobilized-trypsin column; the tryptic fragments were then separated by reversed-phase HPLC. The peptide separation was found to be identical to separation of a sample which had undergone conventional enzymatic protein digestion in solution. Digestion of BSA by the immobilized-trypsin column decreased with increasing flow rate of the solution through the column, and 1.0 muL min(-1) was found to be the optimum flow rate for on-line protein digestion with our system. It was also found that the sample required pretreatment with urea before injection, because of a change in the properties of the protein in the presence of urea, and the immobilized-trypsin column lost its function in the presence of acetonitrile. This on-line proteomics system enables simple and rapid protein digestion and was successfully applied to partially micro two-dimensional (2D) chromatographic separation of proteins.