Atmospheric pressure chemical ionization (APCI) mass spectrometry was investigated as a new method for analysis of a mixture of triglycerides separated by reverse-phase high-performance liquid chromatography. A mixture of homogeneous (monoacid) triglyceride standards containing fatty acids with zero to three double bonds was analyzed to demonstrate the quality of mass spectra obtained by using the APCI interface. The mass spectra showed that minimal fragmentation occurs, resulting primarily in diglyceride [M-RCOO]+ ions and [M + 1]+ protonated molecular ions. The degree of unsaturation within the acyl chains had a marked effect on the proportion of diglyceride ions vs. the [M + 1]+ ions formed in the APCI source. The mass spectra of triglycerides containing fatty acids with two or three double bonds showed predominantly protonated triglyceride ions, with diglyceride peaks representing 13 to 25% of the base peak. The triglycerides containing singly unsaturated fatty acids gave diglyceride ions as the base peak, and [M + 1]+ ions with an intensity 20 to 28% that of the base peak. Only diglyceride ions were observable in the spectra of triglycerides containing saturated fatty acids.