AbstractFourier transform infrared (FTIR) microspectroscopy has been applied to a study of prostate cancer cell lines derived from different metastatic sites and to tissue from benign prostate and Gleason‐graded malignant prostate tissue. Paraffin‐embedded tissue samples were analysed by FTIR, after mounting onto a BaF2 plate and subsequent removal of wax using Citroclear followed by acetone. Cell lines were analysed as aliquots of cell suspension held between two BaF2 plates. It was found that the ratio of peak areas at 1030 and 1080 cm−1, corresponding to the glycogen and phosphate vibrations respectively, suggests a potential method for the differentiation of benign from malignant cells. The use of this ratio in association with FTIR spectral imaging provides a basis for estimating areas of malignant tissue within defined regions of a specimen. Initial chemometric treatment of FTIR spectra, using the linear discriminant algorithm, demonstrates a promising method for the classification of benign and malignant tissue and the separation of Gleason‐graded CaP spectra. Using the principle component analysis, this study has achieved for the first time the separation of FTIR spectra of prostate cancer cell lines derived from different metastatic sites. Copyright © 2003 John Wiley & Sons, Ltd.