We have adapted the low-frequency ESR spectrometer, designed and built by H.J. Halpern, to the physiologic needs of organ preparations operating at 250 MHz. Initial studies have allowed us to detect nitroxides in an isolated perfused heart. These in situ measurements were made with nitroxides specifically designed to mimic the lipophilic nature of 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and 2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxyl (DMPO-OH). These spin labels provided information about the influence of dynamic factors of the heart, such as flow rate, different cell populations and unequal distribution between compartments on our ability to conduct and interpret spin trapping experiments. They also clarified the sacrifice in sensitivity involved in operating at the lower frequencies. To deal with this later problem, we have increased the sensitivity of the spin trapping method by synthesizing a family of 15N- and deuterium-containing DMPO analogs and by determining their ability to spin trap free radicals generated by the model superoxide system of xanthine/xanthine oxidase. Finally, since activated neutrophils are one of the few cells known to generate free radicals as part of their physiologic function, we used these phagocytic cells, as a source of superoxide.