Stable isotope probing (SIP) is a method used for labeling uncultivated microorganisms in environmental samples or directly in field studies using substrate enriched with stable isotope (e.g., (13)C). After consumption of the substrate, the cells of microorganisms that consumed the substrate become enriched in the isotope. Labeled biomarkers, such as phospholipid-derived fatty acid (PLFA), ribosomal RNA, and DNA can be analyzed with a range of molecular and analytical techniques, and used to identify and characterize the organisms that incorporated the substrate. The advantages and disadvantages of PLFA-SIP, RNA-SIP, and DNA-SIP are presented. Using examples from our laboratory and from the literature, we discuss important methodological considerations for a successful SIP experiment.