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This paper demonstrates the use of attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy to detect microbial metabolic products on carbonate mineral surfaces. By creating an ATR-FTIR spectral database for specific organic acids using ATR-FTIR spectroscopy we were able to distinguish metabolic acids on calcite surfaces following Escherichia coli growth. The production of these acids by E. coli was verified using high-performance liquid chromatography with refractive index detection. The development of this technique has allowed us to identify microbial metabolic products on carbonate surfaces in nutrientlimited cave environments.Molecular phylogenetic analysis using the 16S rRNA gene sequence has revolutionized microbiology, allowing investigators to create comparative and evolutionary phylogenies for microbial species (49, 50). Phylogenetic placement also allows metabolic inference for the activity of previously uncultivated species within the environment, dramatically changing our understanding of microbial ecology and the role of microbial species within the biosphere (3, 36, 41). While metabolic inference is a valuable tool for estimating metabolic activity, specific metabolic responses to environmental conditions are harder to estimate, even for closely related species (1,24). To overcome such limitations, investigators can employ a number of different techniques to relate identified microbial phylotypes to environmental metabolic activities, including the use of isotopic probing and quantitative mRNA and metagenomic analyses (43).