beta-cyanoalanine nitrilase activity is shown to exert appreciable control over flux through the plant cyanide (CN) detoxification pathway to asparagine. Arabidopsis overexpressing the -cyanoalanine nitrilase pinA from Pseudomonas fluorescens displayed an increased tolerance to toxic levels of exogenous CN.Plants naturally produce cyanide (CN) which is maintained at low levels in their cells by a process of rapid assimilation. However, high concentrations of environmental CN associated with activities such as industrial pollution are toxic to plants. There is thus an interest in increasing the CN detoxification capacity of plants as a potential route to phytoremediation. Here, Arabidopsis seedlings overexpressing the Pseudomonas fluorescens -cyanoalanine nitrilase pinA were compared with wild-type and beta-cyanoalanine nitrilase knockout line (Atnit4) for growth in the presence of exogenous CN. After incubation with CN, PfpinA seedlings had increased root length, increased fresh weight, and decreased leaf bleaching compared with wild-type, indicating increased CN tolerance. The increased tolerance was achieved without an increase in beta-cyanoalanine synthase activity, the other enzyme in the cyanide assimilation pathway, suggesting that nitrilase activity is the limiting factor for cyanide detoxification. Labeling experiments with [C-13]KCN demonstrated that the altered CN tolerance could be explained by differences in flux from CN to Asn caused by altered beta-cyanoalanine nitrilase activity. Metabolite profiling after CN treatment provided new insight into downstream metabolism, revealing onward metabolism of Asn by the photorespiratory nitrogen cycle and accumulation of aromatic amino acids.