Purification protocols for elastin generally result in greatly damaged elastin fibers and this likely influences the biological response. We here describe a novel protocol for the isolation of elastin whereby the fibers stay intact, and introduce the term "elastin fiber" for intact elastic fibers with elastin as their sole component. As opposed to elastic fibers, elastin fibers do not contain any microfibrils or associated molecules. Elastin fibers were isolated from equine elastic ligaments according to various protocols and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, amino acid quantification, immunofluorescence assay, transmission/scanning electron microscopy, and cellular reactivity in vivo. The optimal protocol comprised several extraction steps and trypsin digestion. Elastin fibers were free of contaminants and had a smooth, regular appearance. The cellular response to purified, intact elastin fibers was different in comparison with purified, but affected, fibers and to contaminated fibers. Intact fibers consisting only of elastin may be important for both fundamental and applied research, for example, tissue engineering, which need well-defined preparations to study the cellular biological effect of individual components.