A series of spin-labeled primary amine derivatives, namely 2,2,6,6-tetramethyl-piperidinyl-1-oxyl-4-amidoalkylamines with varying alkyl chain lengths have been synthesized. The spin-labeled primary amine with tetramethylene or a pentamethylene chain covalently modifies human plasma fibronectin with a stoichiometry of 0.97 to 1.0 (probe-to-subunit) in the presence of coagulation factor XIIIa. The labels with one or two methylene chains similarly modify fibronectin, but with a stoichiometry of only 0.3 to 0.4 per subunit. The spin-labeled primary amine with a trimethylene chain does not label fibronectin. The labeling site appears to be the glutamine-3 residue at the amino-terminal region of fibronectin. Electron spin resonance studies show that the bound labels are partially immobilized with an effective rotational correlation time of 0.4 to 0.6 nsec. The spin-labeled primary amine with tetramethylene chain also is shown to covalently incorporate bee venom melittin in the presence of guinea pig liver transglutaminase. The synthesis of the various spin-labeled primary amines and their applications in the study of structure and dynamics of different proteins and peptides are discussed. The observations from this study suggest that these spin-labeled primary amines potentially have wide application as structural probes.