Although crude acids are minor constituents in petroleum, they have significant implications for crude oil geochemistry, corrosion, and commerce. We have previously demonstrated that a single positive-ion electrospray ionization (ESI) high-field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) experiment can resolve and identify 3000 chemically different elemental compositions of bases (basic nitrogen compounds) in a crude oil. Here, we show that negative-ion ESI high-field FT-ICR MS can selectively ionize and identify naphthenic acids without interference from the hydrocarbon background. When combined with prechromatographic separation, ESI FT-ICR MS reveals an even more detailed acid composition. An average mass resolving power, m/Deltam(50%) greater than or equal to 80 000 (Deltam(50%) is mass spectral peak full width at half-maximum peak height) across a wide mass range (200 < m/z < 1000), distinguishes as many as 15 distinct chemical formulas within a 0.26 Da mass window. Collectively, more than 3000 chemically different elemental compositions containing O-2, O-3, O-4, and O2S, O3S, and O4S were determined in a South American heavy crude. Our data indicates that the crude acids consist of a mixture of structures ranging from C-15-C-55 with cyclic (1-6 rings) and aromatic (1-3 ring) structures. The acid composition appears to be simpler than that of the corresponding hydrocarbon analogues.