A recently developed HPLC technique has been used to identify 10 major monomers from potassium hydroxide hydrolysis and transesterification of lime cutin, revealing 16-hydroxy-10-oxo-hexadecanoic and 10,16-dihydroxyhexadecanoic acids as the major constituents. Solid-state 13CNMR spectroscopy has also been used to examine the unreacted residues following hydrolytic treatment. For transesterification with methanolic boron trifluoride, an alternative protocol using MS has been developed. Using HPTLC to separate epoxy and hydroxy fatty esters, and making a series of trimethylsilyl ether derivatives, the monomeric products have been subjected to analysis by GC-CI-mass spectrometry. Although GC cannot discriminate between positional isomers of oxo and dihydroxy fatty acids, the parent ions and fragmentation patterns obtained with CI-mass spectrometry allow definitive identification of each isomer and reveal five new constituents of the cutin biopolymer. Both the methodology and the monomeric structures are compared with prior reports for citrus fruit cuticle; implications for the molecular architecture and biosynthesis of the lime cutin polymer are also discussed.