Tuberin (I) is shown to derive from glycine by way of tetrahydrofolate metabolism and from tyrosine. Glycine is incorporated into the N-formyl group of tuberin with (partial) stereospecific retention of the 2 pro-S proton; there is also some non-stereospecific loss of deuterium label from [2-¹³C, 2-²H₂]glycine on incorporation into the C₁ units present in tuberin. [2-¹⁴C]-threo-3-Hydroxytyrosine [as (7)] is only incorporated into tuberin after degradation; exclusive labelling of the two C₁ units was observed. [2-³H]Octopamine [as (6)] is a poor precursor for tuberin. Tuberin (1) is a metabolite of Streptomyces amakusaensis. Its structural resemblance to some naturally occurring isonitriles, e.g. xanthocillin-X-monomethyl ether (2), suggested that knowledge of the biosynthesis of tuberin (1) might provide insight into the intriguing mystery of microbial isonitrile biosynthesis. We have also used tuberin as a convenient substrate for studying aspects of stereochemistry associated with microbial C₁-tetrahydrofolate metabolism. Some of our results have appeared in preliminary form.