The quantification of choline-containing compounds (Cho) in breast tumors by proton MR spectroscopy (1H-MRS) has been of great interest because such compounds have been linked to malignancy. In this study, an internal reference method for the absolute quantification of Cho metabolite in malignant breast tumors was presented using a clinical 1.5 T scanner. We performed in vitro measurements to examine the accuracy of absolute quantification using four phantoms of known choline chloride concentrations. There was a high correlation between the calculated concentrations by MRS and the known concentrations (r2 > 0.98). We applied the technique to in vivo breast study conducted on 45 patients with biopsy-confirmed breast cancer. After T1 and T2 relaxation times were corrected, the Cho levels in this work had a range of 0.76-21.20 mmol/kg from 34 MR spectra of 32 patients with malignant breast lesions. This result was rather consistent with the previously published value (i.e., 1.38-10 mmol/kg, Bolan et al. in Magn Reson Med 50:1134-1143, 2003). Therefore, we conclude that the internal method using the fully relaxed water as a reference could be used for quantifying Cho metabolite accurately in breast cancer patients using a clinical 1.5 T scanner.