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The use of 15N techniques allows for the quantitative evaluation of N2 fixation and distribution and their impact on the N balance in various soil‐plant systems. The A‐value approach was used in this investigation to assess N2 fixed at various growth stages in fieldgrown soybean [Glycine max (L.) Merrill] cv. Chippewa in a Typic Eutrocrepts soil. At physiological maturity (R7), the amount of N derived from fixation (Ndfa) was 102 kg/ha, equivalent to 47% of total N assimilated, while the contributions from soil (Ndfs) and 15N‐labelled fertilizer (Ndff) accounted for 50 and 3%, respectively. Up to growth stage V6, which occupied half of the total duration of growth, Ndfa was less than 5% of N2 fixed by physiological maturity. A rapid increase in Ndfa occurred from R1 onwards, and during the reproductive stages (R1‐R4), which spanned less than one‐third of the total duration of growth, this represented about 45% of total Ndfa. An almost equal portion of N (approximately 43%) was fixed from pod‐filling (R5) to physiological maturity (R7), a period slightly more than one‐fifth of the total duration of growth. Therefore, substantial N2 fixation occurred during periods of active sink development and contributed more than 65% of the plant's N accrued during pod fill (R3‐R7). Nitrogen assimilated between R3 and R7 (when N2 fixation was high) seemed to be the predominant source of N for pod development. Thus there was a greater contribution from fixed N (55%) than soil N (43%) in pods and seeds at the end of R7. After grain removal, it was estimated that the growth of cv. Chippewa in this soil led to a net soil depletion of 54 kg N/ha.

Results of field experiments using partial substratum labelling techniques are presented. These show that inorganic 15N application to soil just prior to sowing, the addition of 15N together with sucrose, incorporation of a 15N-labelled plant material into soil, as well as the 15N remaining in soil following the application of inorganic fertilizer to a previous crop provided adequate levels of 15N for field experiments to estimate N2 fixation in soybean and faba beans. These methods may be suitable for quantifying associative N2 fixation, especially if the experimental variability is low, or where N2 fixation is high. Proper site selection is therefore important. Under the conditions of the experiments reported in this paper, however, the sensitivity of detection of N2 fixation was low and could not estimate N2 fixed in plants in which the percentage of N derived from fixation was below 10–15%. The selection of the appropriate reference on standard, i.e., non-N2-fixing, crop and its judicious use in field experiments is crucial to the methodology for quantitatively measuring the amount of N2 fixed by the above methods. The most important factors in the selection of reference crops are absence of N2-fixing activity, similarity in feeding or rooting pattern of the non-N2-fixing and fixing crop, comparable growth period for reference and fixing crop, relative effect of environment on the two crops, and cropping pattern employed. In associative N2 fixation studies, an uninoculated plant in soil devoid of associative N2-fixing microorganisms would provide an ideal reference crop for an inoculated plant.

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