We correlated the in vivo 1H Magnetic Resonance Spectroscopy (MRS) concentration of the choline peak (CHO) with in vitro chemical measures of choline-containing compounds and a histological grade of cellularity in 18 patients with neoplastic and infectious brain lesions. Gas-chromatography-mass-spectrometry (GCMS) was used to measure the concentrations of free choline (Cho), glycerophosphocholine (GPCho), phosphocholine (PCho) and phosphatidylcholine (PtdCho) from biopsies in the same area where MRS was performed. Cellular density, free Cho, PCho and GPCho were the strongest determinants of 1H MRS CHO while PtdCho was not. Just as the 1H MRS 2.0 peak reflects both the concentration of n-acetyl-l-aspartate and neuronal density, the 1H MRS 3.2 peak reflects the concentration of water-soluble choline-containing compounds and cellular density.