Phosphinothricyl-alanyl-alanine (Pt tripeptide (Ptt), bialaphos) is a metabolite produced by Streptomyces viridochromogenes and Streptomyces hygroscopicus. It contains the unique phosphinoamino acid phosphinothricin (Pt), which after cleavage from Ptt is active as an inhibitor of glutamine synthetase. We have isolated three enzymes that assemble the building block of the Ptt peptide backbone in a nonribosomal mechanism. The first enzyme, named Ptt-synthetase I (PTTS I), activates N-acetyldemethylphosphinothricin (AcDMPt) as adenylate and thioester. Pt is not activated. PTTS I can also activate N-acetylphosphinothricin (AcPt) or N-acetylglutamate as structural analogues of AcDMPT. Native PTTS I has an estimated size of 62 kDa whereas the denatured form displays a size of 76 kDa. Immunoblot analysis and determination of its N-terminal protein sequence revealed that PTTS I is identical with the gene product of phsA. The phsA gene was previously identified near the Pt-resistance gene pat in the Ptt biosynthesis gene cluster in S. viridochromogenes. Besides PTTS I, two alanine-activating enzymes (PTTS II/III) were partially purified from S. viridochromogenes with estimated native sizes of ca. 120 kDa (enzyme 1) and ca. 140 kDa (enzyme 2). Both enzymes bind alanine as a thioester via the corresponding adenylate. Level of PTTS II/III and product formation were correlated with each other in several different strains of S. viridochromogenes. These results indicate that Ptt is synthesized by three peptide synthetases, each activating one single amino acid. The data also confirm previous genetic data, which suggest that AcDMPt-Ala-Ala is the precursor of Ptt.