The effect of nitrate on N(2) fixation and the assimilation of fixed N(2) in legume nodules was investigated by supplying nitrate to well established soybean (Glycine max L. Merr. cv Bragg)-Rhizobium japonicum (strain 3I1b110) symbioses. Three different techniques, acetylene reduction, (15)N(2) fixation and relative abundance of ureides ([ureides/(ureides + nitrate + alpha-amino nitrogen)] x 100) in xylem exudate, gave similar results for the effect of nitrate on N(2) fixation by nodulated roots. After 2 days of treatment with 10 millimolar nitrate, acetylene reduction by nodulated roots was inhibited by 48% but there was no effect on either acetylene reduction by isolated bacteroids or in vitro activity of nodule cytoplasmic glutamine synthetase, glutamine oxoglutarate aminotransferase, xanthine dehydrogenase, uricase, or allantoinase. After 7 days, acetylene reduction by isolated bacteroids was almost completely inhibited but, except for glutamine oxoglutarate aminotransferase, there was still no effect on the nodule cytoplasmic enzymes. It was concluded that, when nitrate is supplied to an established symbiosis, inhibition of nodulated root N(2) fixation precedes the loss of the potential of bacteroids to fix N(2). This in turn precedes the loss of the potential of nodules to assimilate fixed N(2).