In addition to some usual fatty adds, the seed oil of Jodina rhombifolia (Santalaceae) contains nine acetylenic fatty acids [9-octadecynoic acid (stearolic acid) (1.1%), trans-10heptadecen-8-ynoic acid (pyrulic acid) (20.1%), 7-hydroxytrans-l(~heptadecen-8,ynoic acid (2.3%), trans-10,16-heptadecadien-8-ynoic acid (0.7%), 7-hydroxy-trans-10,16-hepta-decadien~-ynoic acid (0.1%), trans~ll~actadecen-~ynoic acid (ximenynic acid) (20.3%), 8-hydroxy-trans-ll-octadecen-~ ynoic acid (12.2%), trans-ll,17-octadecadien-9-ynoic acid (1.5%), 8-hydroxy-trans-ll,17-octadecadien-9-ynoic acid (1.3%), 9-hydroxystearic acid (<0.1%) and 9,10-epoxystearic acid (0.7%)]. The fatty acids have been analyzed by gas chromatography/mass spectrometry of their methyl ester and 4,4-dimethyloxazoline derivatives. The hydroxy fatty acid methyl esters have been examined also as trlmethyl-sUyl ethers. Furthermore, the fatty acid methyl esters (FAME) have been fractionated according to their polarity (FAMF_~A: nonhydroxy; FAMF~B: hydroxy fatty acids) and to their degree of unsaturation (FAME-A1/A2; FAME-BI/B2) by preparative thin-layer chromatography and argentation chromatography, respectively. All of these fractions have been analyzed by ultraviolet and infrared spectroscopy, and the fractions FAME-A and FAME-B have been analyzed further by nuclear magnetic resonance (1H, 13C, 2D H/C, attached proton test) spectroscopy and gas chromatography/mass spectrometry.KEY WORDS: Acetylenic fatty acids, epoxy fatty acids, gas chromatography/mass spectrometry of 4,4-dimethyloxazoline derivatives of acetylenic fatty acids, hydroxy fatty acids, Jod/na rhombifolia, NMR spectroscopy, Santalaceae.