The mechanism of the two-layer method (TLM) of pancreas preservation is unclear. Facilitating oxygen diffusion into preserved pancreas has been suggested, but direct measurements of tissue pO(2) have yielded conflicting results. The degree of penetration of perfluorocarbon (PFC) into the pancreas during TLM storage is unknown. Segments of porcine pancreas (7.5 cm in length) were preserved either in University of Wisconsin solution (UW) alone (n = 6) or in TLM for 24 h (n = 6). Pancreatic samples were analyzed using Varian INOVA 9.4 T MR scanner. External PFC standard was introduced for quantification. Four consecutive transverse images of 4 mm thickness were obtained using a spin-echo sequence. (19)Fluorine magnetic resonance spectroscopy ((19)F MRS) was performed with the same parameters except with more averages. MR data were confirmed by headspace chromatography. PFC standard was readily detected in (19)F MR images. There was no signal from pancreas in (19)F MR images following either UW or TLM storage. (19)F MR spectra typical of PFC were not obtained from either UW- or TLM-preserved pancreas with nonlocalized (19)F MRS. Mean concentration of PFC in TLM pancreas measured by head space chromatography was 0.011 nl/g (SD ± 0.006), not significantly different from background concentration (0.012 nl/g, SD ± 0.006) in UW pancreas (p = 0.42). There was no evidence of penetration of PFC into pancreas tissues investigated either by MR or chromatography in organs preserved at hypothermia by TLM, and mechanisms of TLM remain speculative.