Immobilization of laboratory animals is a basic requirement for experimental in vivo NMR measurements. The effect of single and repeated isoflurane anesthesia on proton NMR relaxation times T1 and T2 in rat liver was studied. Furthermore, physiological monitoring was performed to evaluate the influence of isoflurane anesthesia (up to 2 hr) on biological parameters. Neither single nor repeated isoflurane application over the observed time produce relevant alterations of physiological parameters or relaxation times, compared with untreated control groups. Therefore, we conclude that isoflurane anesthesia is appropriate for in vivo NMR investigations, especially of the liver.