Although nanoelectrospray offers low sample consumption and improved detection limits for proteomic studies, it is currently a low throughput technique because of its tedious single-sprayer alignment procedures. Here, a fully automated nanoelectrospray system for proteomic analyses with mass spectrometric detection is described and characterized. This infusion system aspirates samples from a 96-well plate using disposable pipette tips and then delivers samples to an ESI Chip. This chip is a fully integrated monolithic device consisting of a 10 x 10 array of nozzles. The automated nanoelectrospray system is easily controlled with software that allows the user simply to select the number of samples to analyze, the volume of sample to aspirate, and the spray voltage and head pressure to apply. The system offers all the advantages of conventional nanoelectrospray plus automated, high throughput analyses with no carryover. The high degree of reproducibility and lack of carryover of the system are demonstrated here using a Micromass LCT time-of-flight mass spectrometer with the infusion of six tryptic digests through all 100 nozzles of a chip. The effects of ammonium acetate and sodium dodecyl sulfate are discussed, as well as the system's ability to spray a variety of different solvents. The spray stability of whole cytochrome c in 99.9% water with 0.1% acetic acid over a 15-min period was determined to be 5.06%. Using a Thermo Finnigan LCQ Deca ion trap and SEQUEST search software, 2 fmol/microL myoglobin and 1 fmol/microL cytochrome c digests were unambiguously identified via infusion analyses. Finally, protein spots excised from two-dimensional gels of yeast and E. coli crude cell extracts were identified with the fully automated nanoelectrospray system coupled to an LCQ.