The low molecular weight "blue" copper protein, azurin, has been purified from Pseudomonas putida (NCIB 9869) to homogeneity using various chromatographic techniques including reverse-phase HPLC. The amino acid sequence of the N-terminus of the reduced and carboxymethylated protein yielded a single sequence corresponding to AECKV. The complete sequence, comprising 128 amino acid residues with a C-terminal sequence corresponding to TVTLK, was determined from the peptides obtained from a Staphylococcus aureus V8 digest of the protein and confirmed using peptides obtained following cyanogen bromide and endoprotease Asp-N digests. The amino acid sequence contained three cysteine residues at positions 3, 26, and 112, was devoid of tryptophan, and showed closest similarity (90% identical residues) to the previously determined sequence of azurin isolated from Pseudomonas fluorescens biotype B [Ambler, R.P. (1971) in Developpements Recents Dans L'Etude Chimique De La Structures Des proteins (Preverio, A., Pechere, J.-F., and Coletti-Preverio, M.-A., Eds.), pp. 289-305, INSERM, Paris]. Examination of the complete sequence indicated P. putida azurin contained unique Asp and Ala residues at positions 19 and 21, respectively, that have not been found in any other azurin sequence.