Specific inhibitors of cytochrome P-450, a well-known oxidative enzyme, have been successfully used to identify less oxidized biosynthetic intermediates of secondary metabolites. To explore their utility in biosynthetic studies of chaetoglobosin A (1), we treated Chaetomium subaffine with the cytochrome P-450 inhibitor metyrapone. Extraction and purification of the mycelium yielded four new compounds, prechaetoglobosins I-IV. These compounds were characterized as less oxidized derivatives of 1 through molecular formula determination, mass spectrometry (noting the indolylmethyl fragment m/z 130), and extensive NMR analyses (1D and 2D), which revealed differences in oxidation levels at positions C-6, C-7, C-19, and C-20 compared to 1. Quantitative HPLC analysis demonstrated that metyrapone was more effective than other inhibitors (e.g., S-3307D) at accumulating these less oxidized compounds, with prechaetoglobosins I, II, and III showing 25.5, 3.8, and 5.6-fold higher levels than the control. These new metabolites are plausible precursors of 1, and their structures enabled the proposal of a biosynthetic pathway for 1. The isolation of prechaetoglobosin I (2) suggests the hypothetical hexacene (7), a previously proposed non-oxidized form, undergoes [4+2] cycloaddition to form 2.