The intra-orbital lacrimal gland (Harderian gland, or HG) of the female rat was studied by magnetic resonance imaging (MRI) to evaluate whether MRI can be used to visualize the gland in vivo and localized-1H-spectroscopy detect its lipid content. The results were correlated with post-mortem anatomical sections, and with light and electron microscopy. On MRI, HG presented as a mass located between the ocular bulb and the orbit. In strongly T2W sequences the secretory structures had a reduced signal while intraparenchymal connective tissue was visible. T2-quantitative maps values of HG (60.12 +/- 8.15 ms, mean +/- SD) were different from other tissues (i.e. muscular tissue, T2 = 44.79 +/- 3.43 ms and olfactory bulb, T2 = 79.26 +/- 4.25 ms). In contrast-enhanced-MRI, HG had a signal-intensity-drop of 0.074 +/- 0.072 (mean +/- SD), after injection of AMI-25, significantly different from the muscle (0.17 +/- 0.10). Localized MRI spectra gave a large part of the signal originating from fat protons, but with a significant percentage from water protons. At light and electron microscopy the lipid deposition appeared to be composed of low-density material filling a large part of the cytoplasm, and the porphyrin aggregates were easily recognizable. The data demonstrate that an in vivo study of the HG was feasible and that high-field MRI allowed analysis of the gross anatomy detecting the lipid content of the gland.