Raman microspectroscopy is an emerging tool to analyze the molecular and isotopic composition of single microbial cells. It can be used to achieve an in situ understanding of metabolic processes. Due to the low sensitivity of the Raman effect, surface-enhanced Raman scattering (SERS) is utilized to enhance the Raman signal. The SERS spectra of bacteria are usually characterized by a pronounced band at around 730 cm(-1), which is assigned to glycosidic ring vibrations or to adenine or even to CH2 deformation in different studies. In order to clarify the origin of this band, we employed a stable isotope approach and performed a SERS analysis of Escherichia coli bacteria using in situ prepared Ag nanoparticles. The cells were grown on unlabeled ((12)C, (14)N) and labeled ((13)C, (15)N) carbon and nitrogen sources in different combinations. The SERS band of the stable isotope labeled microorganisms showed a characteristic red-shift in the SERS spectra, which solely depends on the isotopic composition. It was therefore possible to confidently assign this band to adenine-related compounds. Furthermore, by utilizing the fingerprint area of single-cell SERS spectra as the input for the principal component analysis, one can clearly differentiate between E. coli bacteria incorporating different stable isotopes.