An optimized sequencer program with a cycle time of 38 min which is specifically tailored for analysis using polyvinylidene difluoride (PVDF) membranes has been developed. The program was developed using a pulsed liquid-phase instrument which was converted to gas-phase acid delivery. Gas-phase acid delivery minimized sample extraction from PVDF membranes and improved tryptophan yields in at least some cases. Other modifications which contributed to reliable high sensitivity sequencer performance included use of a Blott cartridge, substitution of ethyl acetate:heptane (1:1, v/v) instead of butyl chloride as the extraction solvent, use of a modified 100-microliters injection loop with an internal restrictor to reliably inject nearly 90% of the sample, and an HPLC gradient which resolved tryptophan from diphenylurea. These shortened cycle times were achieved at the conventional gas-phase reaction temperature. A slight increase in lag or carryover at prolines was compensated by reduced background from nonspecific acid cleavage which facilitated extended and/or high sensitivity sequencing of large proteins. Reproducible high initial and repetitive cycle yields were obtained with a wide range of experimental peptides which were electroblotted from either 1D or 2D polyacrylamide gels onto high retention PVDF membranes. Initial yields of the majority of the experimental samples analyzed with this program were less than 5 pmol. In addition, most samples with initial yields below 1-2 pmol yielded sufficient sequence information to identify the protein by comparison to protein sequence data-bases or to design oligonucleotide probes.