Summary Nitrilase (E.C. 3.5.5.1) cloned from Arabidopsis thaliana converts indole‐3‐acetonitrile to the plant growth hormone, indole‐3‐acetic acid in vitro. To probe the capacity of this enzyme under physiological conditions in vivo, the cDNA PM255, encoding nitrilase II, was stably integrated into the genome of Nicotiana tabacum by direct protoplast transformation under the control of the CaMV‐35S promotor. The regenerated plants appeared phenotypically normal. Nitrilase II was expressed, based on the occurrence of its mRNA and polypeptide. The enzyme was catalytically active, when extracted from leaf tissue of transgenic plants (specific activity: 25 fkat mg−1 protein with indole3‐acetonitrile as substrate). This level of activity was lower than that found in A. thaliana, and this was deemed essential for the in vivo analysis. Leaf tissue from the transgenic plants converted 1‐[13C]‐indole‐3‐acetonitrile to 1‐[13C]‐indole‐3‐acetic acid in vivo as determined by HPLC/ GC‐MS analysis. Untransformed tobacco was unable to catalyze this reaction. When transgenic seeds were grown on medium in the absence of indole‐3‐acetonitrile, germination and seedling growth appeared normal. In the presence of micromolar levels of exogenous indole‐3‐acetonitrile, a strong auxin‐overproducing phenotype developed resulting in increased lateral root formation (at 10 µM indole‐3‐acetonitrile) or stunted shoot growth, excessive lateral root initiation, inhibition of root out‐growth and callus formation at the root/shoot interface (at 100 µM indole‐3‐acetonitrile). Collectively, these data prove the ability of nitrilase II to convert low micromolar levels of indole‐3‐acetonitrile to indole‐3‐acetic acid in vivo, even when expressed at subphysiological levels thereby conferring a high‐auxin phenotype upon transgenic plants. Thus, the A. thaliana nitrilase activity, which exceeds that of the transgenic plants, would be sufficient to meet the requirements for auxin biosynthesis in vivo.