Although the d-glucarate degradation pathway is well characterized in Escherichia coli, genetic and biochemical information concerning the alternative pathway proposed in Pseudomonas species and Bacillus subtilis remains incomplete. Acinetobacter baylyi ADP1 is a Gram-negative soil bacterium possessing the alternative pathway and able to grow using d-glucarate as the only carbon source. Based on the annotation of its sequenced genome (1), we have constructed a complete collection of singlegene deletion mutants (2). High throughput profiling for growth on a minimal medium containing d-glucarate as the only carbon source for ∼2450 mutants led to the identification of the genes involved in d-glucarate degradation. Protein purification after recombinant production in E. coli allowed us to reconstitute the enzymatic pathway in vitro. We describe here the kinetic characterization of d-glucarate dehydratase, d-5-keto-4-deoxyglucarate dehydratase, and of cooperative α-ketoglutarate semialdehyde dehydrogenase. Transcription and expression analyses of the genes involved in d-glucarate metabolism within a single organism made it possible to access information regarding the regulation of this pathway for the first time.