Abstract We report here the results of our study on speciation and isotope ratios of N in fluid inclusions preserved in 3.5 Ga hydrothermal deposits (silica dikes and quartz veins) from the North Pole area of the Pilbara Craton, Western Australia. Crush-leach analysis and Raman microspectrometry revealed that N within the fluid inclusions exists as N2 and NH4+. A negative correlation between the SO42−/Na+ and 40Ar/36Ar ratios of the fluid inclusions suggests mixing of two end-members; hydrothermal fluid with low SO42−/Na+ and high 40Ar/36Ar ratios, and 3.5 Ga seawater with high SO42−/Na+ and low 40Ar/36Ar ratios. Values of δ15NN2 from the hydrothermal component vary over a considerable range (− 3.0 to + 3.7‰), and those of the seawater component are well within this range (i.e., − 0.7 to − 0.2‰). This suggests that the isotope ratio of N2 dissolved in the 3.5 Ga seawater would have been − 0.7 to − 0.2‰. Since isotope fractionation between N2 in the atmosphere and N2 dissolved in seawater is minimal, the δ15NN2 value of the 3.5 Ga atmosphere would have been within the range − 2~0‰, which is similar to the δ15NN2 value of the present-day atmosphere (δ15NN2 = 0‰). This study also suggests that the fluid inclusions contain NH4+ that would have been derived from the seawater and/or mantle at 3.5 Ga. Therefore, kerogens in Archean cherts might have been partly derived from biological assimilation of NH4+ in hydrothermal fluids.