ABSTRACT:   Electron Spin Resonance at high magnetic fields (HF ESR) is rapidly developing into a powerful biophysical tool which is uniquely positioned to address complex aspects of structure and dynamics of proteins, membranes, and macromolecular assemblies at molecular level. The source of the contemporary resurgence of interest in ESR as a biophysical tool is that there are, broadly speaking, three large groups of problems, which can be approached with ESR methods but cannot easily be studied by traditional structural techniques: (1) structure and dynamics of large molecular weight proteins in solution; (2) membrane and membrane-associated proteins: structure, location with respect to the membrane, side-chain dynamics, and interactions with other membrane components or DNA's and RNA's; (3) fast conformational transitions of proteins and RNA's in solution, protein folding and re-folding. The focus of this review is mainly on HF ESR spin-labeling techniques, primarily via nitroxide spin labels, as this method is the most flexible and is even applicable to proteins which are otherwise ESR-silent. We start with physical aspects of ESR of nitroxide spin labels at high magnetic fields in order to categorize the characteristic information that can be gained from such experiments. Then we describe practical applications of spin-labeling HF ESR to study structure and dynamics of complex biophysical systems. We also discuss several details of the ESR motional theory based on the stochastic Liouville equation (SLE) that are relevant to nitroxide line shape analysis in order to appreciate both the information available from and the limitations of the method. Particular emphasis is given to multifrequency HF ESR methods in studies of spin-labeled membranes and biopolymers. Finally, we review the use of HF ESR in applications to molecular structure including distance measurements, determination of molecular orientations from ESR of ordered samples, and structural studies based on g-factor measurements.