Despite considerable attention over the last 25 yr, the importance of early protein breakdown products to plant nitrogen (N) nutrition remains uncertain. We used rhizosphere injection of 15N-, 13C- and 14C-labelled inorganic N and amino acid (l-alanine), with chase periods from 1 min to 24 h, to investigate the duration of competition for amino acid between roots (Triticum aestivum) and soil microorganisms. We further investigated how microbial modification of l-alanine influenced plant carbon (C) and N recovery. From recovery of C isotopes, intact alanine uptake was 0.2–1.3% of added. Soil microbes appeared to remove alanine from soil solution within 1 min and release enough NH4 + to account for all plant 15N recovery (over 24 h) within 5 min. Microbially generated inorganic or keto acid C accounted for < 25% of the lowest estimate of intact alanine uptake. Co-location of C and N labels appears a reasonable measure of intact uptake. Potential interference from microbially modified C is probably modest, but may increase with chase period. Similarly, competition for l-alanine is complete within a few minutes in soil, whereas NO3 − added at the same rate is available for > 24 h, indicating that long chase periods bias outcomes and fail to accurately simulate soil processes.