- Fourier transform infrared (FTIR) spectroscopy is currently being developed as a new optical approach to the diagnosis and characterization of cell or tissue pathology. The advantage of FTIR microspectroscopy over conventional FTIR spectroscopy in the diagnosis of malignancies is that it facilitates inspection of restricted regions of the cell culture or tissue. In this study, we set out to evaluate FTIR microspectroscopy as a diagnostic tool for identifying retrovirus‐induced malignancies. Our study showed significant and consistent differences between cultures of different types of cells of both mouse and human origin, i.e. primary fibroblast cells (one to two passages in cell culture), fibroblast cell lines and malignant cells transformed by murine sarcoma virus. An impressive decrease in the levels of phosphate and other metabolites was seen in malignant cells compared with primary cells. The levels of these metabolites in the cell lines were significantly lower than in the primary cells but higher than in the malignant cells. In addition, the peak attributed to the PO2− symmetric stretching mode at 1082 cm−1 in primary cells shifted significantly to 1085 cm−1 for the cell line and to 1087 cm−1 for the malignant cells. These differences taken together with differences in the shapes of various bands throughout the spectrum strongly support the possibility of developing FTIR microspectroscopy for the detection and study of malignant—and possibly premalignant—cells.