We used a combined atomic force microscopic (AFM)/fluorescence microscopic technique to study the mechanical properties of individual, electrospun fibrinogen fibers in aqueous buffer. Fibers (average diameter 208 nm) were suspended over 12 microm-wide grooves in a striated, transparent substrate. The AFM, situated above the sample, was used to laterally stretch the fibers and to measure the applied force. The fluorescence microscope, situated below the sample, was used to visualize the stretching process. The fibers could be stretched to 2.3 times their original length before breaking; the breaking stress was 22 x 10 (6)Pa. We collected incremental stress-strain curves to determine the viscoelastic behavior of these fibers. The total stretch modulus was 17.5 x 10 (6)Pa and the relaxed elastic modulus was 7.2 x 10 (6)Pa. When held at constant strain, electrospun fibrinogen fibers showed a fast and slow stress relaxation time of 3 and 55 s. Our fibers were spun from the typically used 90% 1,1,1,3,3,3-hexafluoro-2-propanol (90-HFP) electrospinning solution and re-suspended in aqueous buffer. Circular dichroism spectra indicate that alpha-helical content of fibrinogen is approximately 70% higher in 90-HFP than in aqueous solution. These data are needed to understand the mechanical behavior of electrospun fibrinogen structures. Our technique is also applicable to study other nanoscopic fibers.