Phosphinothricin-tripeptide (PTT, phosphinothricyl-alanyl-alanine) is a natural product antibiotic and potent herbicide that is produced by Streptomyces hygroscopicus ATCC 217051 and Streptomyces viridochromogenes DSM 407362. PTT has attracted widespread interest due to its commercial applications and unique phosphinic acid functional group. Despite intensive study since its discovery in 1972 (see3 for a comprehensive review), a number of steps early in the PTT biosynthetic pathway remain uncharacterized. Here we report a series of interdisciplinary experiments involving the construction of defined S. viridochromogenes mutants, chemical characterization of accumulated intermediates, and in vitro assay of selected enzymes to examine these critical steps in PTT biosynthesis. Our results indicate that early PTT biosynthesis involves a series of heretofore undescribed catalyses, including a highly unusual reaction for carbon bond cleavage. In sum, we define a more complex pathway for early PTT biosynthesis that includes biochemically unprecedented and chemically interesting steps.