We describe assembly and optimization of a continuous flow nanoelectrospray source for high-performance analysis on a routine basis. It is derived from an inJection adaptable Fine Ionization Source ("JaFIS"), previously shown to be durable and easy to use (Geromanos, S.; et al. Rapid Commun. Mass Spectrom. 1998, 12, 551-556) and now modified for maximum sensitivity. Proper design, manufacturing, and quality control of spray needles with specific orifice diameters, in combination with precisely controlled helium backpressure and applied voltage, enable stable flows at 1-2 nL/min. Needle positioning and ion spray potential are hereby exceedingly important, as shifts by 0.5 mm or 25 V, respectively, cause significant reduction in signal strength. In addition to prolonged analysis times, ultralow flows also yield higher sensitivity, the result of an improved "overall ion transfer efficiency" measured to be approximately 5% at 1.6 nL/min. Used in combination with a "microtip" (Erdjument-Bromage, H.; et al. J. Chromatogr. A 1998, 826, 167-181), the optimized JaFIS implements infusion-style ESI-MS at sensitivities approaching capillary LC-MS. Spraying times in excess of 20 h allow for any number of tandem mass spectrometric analysis routines to be performed, and to average thousands of scans in every experiment, thereby further improving sensitivity. This was fully illustrated by extensive analysis of a 2-fmol peptide mixture, in a 2-microL volume, using a multimode MS approach.