Fluorine nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging were examined as noninvasive methods for characterizing antibiotic disposition and pharmacokinetics in vivo. For determination of their utility, a 19F surface coil was constructed and an m-(trifluoromethyl)-containing penicillin V analogue (LY242072; 1) was synthesized. Various concentrations of 1 were injected intravenously into anesthetized rats whose urethras were occluded. The animals were placed on the surface coil, which was tuned to 19F, and then into a 4.7-T, 33-cm bore instrument, in which in vivo measurements of 1 were made on urine excreted into the bladder. At sacrifice, the urine was collected, and antibiotic levels were determined in vitro by both HPLC and high-resolution NMR. The limit of detection of 1 by NMR was 0.7 mg/mL of urine. When compared with standard in vitro quantitative methods using current technology, quantitation by in vivo surface coil NMR is not precise. Magnetic resonance imaging was used to image the bladder at a 35-mm3 voxel resolution with datum collection times of approximately 1 h. The 19F surface coil was used successfully to spectroscopically locate xenobiotic fluorine in the rat thorax. 19F NMR may offer an opportunity for the noninvasive in vivo detection of the distribution of various classes of therapeutic compounds.