In recent years, polyvinylidene difluoride (PVDF) membranes have emerged as the preferred support matrices for N-terminal sequence analysis and related protein chemical methods for several reasons. The vast majority of proteins and large peptides bind strongly to PVDF membranes at high capacity, and PVDF has high chemical resistance compatible with diverse analysis protocols and strategies. Proteins can be readily purified for sequence analysis in the low-microgram to submicrogram range using one-dimensional or two-dimensional polyacrylamide gel electrophoresis (PAGE) followed by high-efficiency electrotransfer to PVDF membranes. Since PAGE is the highest resolution microscale protein separation technique currently available, most proteins of interest can be readily isolated and structurally characterized using these methods, provided that the position is known for the protein of interest on an appropriate one-dimensional or two-dimensional PAGE system. In this article, methods are described for efficient electrotransfer of proteins onto high-retention PVDF membranes and for N-terminal sequence analysis of electroblotted proteins using either a gas-phase sequencer or a biphasic cartridge sequencer with sequencer cycle times of approximately 40 min. Electrotransfer recoveries of about 50 to 80% can usually be obtained for most proteins if the recommended methods are carefully followed. Similarly, by optimizing sequencer performance, substantial sequence information can be obtained when as little as 1 to 10 pmol of sequenceable protein is loaded into the sequencer.