We investigated the microbial pathways of nitrogen (N) loss in an April 2005 transect through the Peruvian oxygen minimum zone (OMZ) at 12°S latitude using short anaerobic incubations with 15N-labeled substrates and molecular-ecological and lipid-biomarker studies. In incubations with 15NH4+, immediate production of 14N15N, but not 15N15N, indicated that N2 was produced by the pairing of labeled 15NH 4+ with in situ 14NO2- via anaerobic ammonium oxidation (anammox). Supporting this finding, we also found anammox-related 16S ribosomal ribonucleic acid gene sequences similar to those previously known from other marine water columns in which anammox activity was measured. We identified and enumerated anammox bacteria via fluorescence in situ hybridization and quantitative polymerase chain reaction and found ladderane membrane lipids specific to anammox bacteria wherever anammox activity was measured by our isotope tracer method. However, in incubations with 15NO3- or 15NO2-, in which denitrification would have been expected to produce 15N15N by pairing of oxidized 15N ions, 15N15N production was not detected before 24 h, showing that denitrification of fixed N to N2 was not taking place in our samples. At the time and locality of our study, anammox, rather than denitrification, was responsible for N2 production in the Peruvian OMZ waters. © 2007, by the American Society of Limnology and Oceanography, Inc.