Fiber mats of a synthetic anionic copolypeptide of l-glutamic acid and l-tyrosine (PLEY) have been produced by electrospinning, and physical, chemical, and biological properties of the fibers have been characterized in vitro. Fibers were obtained from polymer dissolved in water at concentrations of 20-60% (w/v) but not below this range. Applied voltage and spinneret-collector distance were also found to influence polymer spinnability. Oriented fibers were obtained by changing the geometry of the collector. Fiber diameter was measured by scanning electron microscopy (SEM). A common chemical reagent was used to cross-link polymers postspinning. Fiber solubility in aqueous solution varied as a function of cross-linking time. Cationic polypeptides labeled with a fluorescent dye became noncovalently associated with cross-linked fibers, enabling visualization by fluorescence microscopy. Spectroscopy provided information on polymer chain conformation in solution and in fibers. Degradation of cross-linked fibers by different proteases has been demonstrated. Fibroblasts were cultured on cross-linked fiber mats to test basic cytocompatibility. Synthetic polypeptide fiber mats may be useful in applications in medicine, biotechnology, and other areas.