The presence of naphthenic acids in crude oils is of concern in the petroleum industry due to their corrosivity to refinery units. It is desirable to determine the ring type and carbon number distributions because the corrosivity of naphthenic acids is dependent on the sizes and structures. The characterization of naphthenic acids is also of interest to geochemical studies, particularly migration and biodegradation, and to refinery wastewater treatment for environmental compliance. We have evaluated chemical ionization, liquid secondary ion mass spectrometry (fast ion bombardment), atmospheric pressure chemical ionization (APCI), and electrospray ionization in both positive and negative ion modes for the determination of molecular distribution of acids without derivatization. Negative-ion APCI using acetonitrile as a mobile phase yields the cleanest spectra with good sensitivity among the ionization techniques evaluated. The selectivity of negative-ion APCI for naphthenic acids has also been demonstrated by comparing results for a whole crude oil with those for the isolated acid fraction. APCI also holds a great potential for on-line Liquid chromatography-mass spectrometric (LC/MS) to separate acids by high-performance liquid chromatography (HPLC) followed by mass spectrometric characterization of acids.