Nitrogen (N_2) fixation was investigated at Mound 12, Costa Rica, to determine its spatial distribution and biogeochemical controls in deep-sea methane seep sediment. Using ^(15)N_2 tracer experiments and isotope ratio mass spectrometry analysis, we observed that seep N_2 fixation is methane-dependent, and that N_2 fixation rates peak in a narrow sediment depth horizon corresponding to increased abundance of aggregates of anaerobic methanotrophic archaea (ANME-2) and sulfate-reducing bacteria (SRB). Using fluorescence in situ hybridization coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS), we directly measured ^(15)N_2 uptake by ANME-2/SRB aggregates (n = 26) and observed maximum ^(15)N incorporation within ANME-2-dominated areas of the aggregates, consistent with previous analyses. NanoSIMS analysis of single cells (n = 34) from the same microcosm experiment revealed no ^(15)N_2 uptake. Together, these observations suggest that ANME-2, and possibly physically associated SRB, mediate the majority of new nitrogen production within the seep ecosystem. ANME-2 diazotrophy was observed while in association with members of two distinct orders of SRB: Desulfobacteraceae and Desulfobulbaceae. The rate of N_2 fixation per unit volume biomass was independent of the identity of the associated SRB, aggregate size and morphology. Our results show that the distribution of seep N_2 fixation is heterogeneous, laterally and with depth in the sediment, and is likely influenced by chemical gradients affecting the abundance and activity of ANME-2/SRB aggregates.