Cetocyclinet is a broad spectrum antibiotic belonging to the tetracycline family. Because of its unusual structural features and biological properties, a continuing interest in this antibiotic has developed. Several factors led us to explore its biosynthesis: while the biosynthetic origin of the tetracyclines has been established as polyketide, relatively few of the individual carbon atoms have been confirmed by direct experimentation; the origin of the "starter" unit has been notoriously difficult to establish directly; an uneven efficiency of incorporation of "extender" units has been observed, raising the possibility that the polyketide skeleton might not form in a concerted manner; the application of stable isotope methodology allows direct observation of all carbons; the 2-acetyl moiety is more convenient for examining the "starter" unit origin than the 2-carboxamide moiety of clinically used tetracyclines; and the biosynthetic pathways of Nocardia species have received less study than those of Streptomyces. For incorporation experiments, Nocardia sulphurea (Abbott isolate S1A75B) was grown in a specific medium, with CH313CO2Na added at 96, 120, 144, and 168 hours. The culture was harvested at 240 hours, and N-acetyl cetocycline was isolated and purified. 13C NMR spectra were recorded to analyze isotopic enrichment. Results showed that acetate serves as the "starter" unit, with an alternating enrichment pattern consistent with polyketide theory. Experiments using [1-13C]-labeled propionate resulted in no measurable enrichment of any carbon, consistent with the two C-methyl groups originating from methionine. The starter unit for classical tetracyclines is usually suggested to be malonamyl coenzyme A, and bioisosteric analogy suggests the starter unit for 2-acetyltetracyclines (including cetocycline) should be acetoacetyl coenzyme A, but work published after submission of this paper rules out this hypothesis.