Due to widespread manipulation of nitrogen (N), much research has focused on processes controlling the fate of anthropogenic N in streams. Yet, in a variety of oligotrophic systems, N fixed by periphyton is a significant driver of ecosystem metabolism. Due to difficulties partitioning allochthonous and autochthonous sources, there is limited information regarding how the latter is processed. Autochthonous N may be particularly important in alpine, arid, or polar environments. We test the hypothesis that the availability of remineralized autochthonous N is controlled by connectivity between the hyporheic zone and main channel due to the contrasting biogeochemical functions of benthic autotrophs (including N‐fixing Nostoc) and hyporheic heterotrophs in an intermittent Antarctic stream. There, we collected surface water and hyporheic water concurrently at 4‐6 hour intervals over a 32.5‐hr period during one flow season and opportunistically throughout a second. Hyporheic water had 7 to 30 times greater nitrate‐N concentrations relative to surface water across all flow conditions. In contrast, ammonium concentrations were generally lower, although similar among locations. Additionally, nitrate in hyporheic water was positively correlated with silica, an indicator of hyporheic residence time. A laboratory assay confirmed prior inferences that hyporheic microbial communities possess the functional potential to perform nitrification. Together, these findings suggest that remineralized autochthonous N accumulates in the hyporheic zone even as streamflow varies and likely subsidizes stream N availability—which supports prior inferences from N stable isotope data at this site. These results highlight the importance of hyporheic connectivity in controlling autochthonous N cycling and availability in streams.