<jats:title>Abstract</jats:title> <jats:p> The nucleoside-peptide antibiotic nikkomycin X contains the unusual heterocyclic base 4-formyl-4-imidazoline-2-on. Investigations on the biosynthesis of this base were accomplished in a resting cell system which was optimized in respect to nikkomycin production. <jats:p>Incorporation experiments with [2-<jats:sup>14</jats:sup>C]adenine, [8-<jats:sup>14</jats:sup>C]adenine, [2-<jats:sup>14</jats:sup>C]glycine, [2-<jats:sup>14</jats:sup>C]uracil, [U-<jats:sup>14</jats:sup>C]histidine, and [2-<jats:sup>14</jats:sup>C]histidine revealed that only [2-<jats:sup>14</jats:sup>C ]adenine, [U-<jats:sup>14</jats:sup>C]-and [2-<jats:sup>14</jats:sup>C]histidine were specifically incorporated into nikkomycin X. An incorporation experiment with [2-<jats:sup>13</jats:sup>C]adenine resulted in a 31-fold enrichment of the carbon atom 2 of 4-formyl-4-imidazoline-2-on. These results show that the heterocyclic base of nikkomycin X is a product of the histidine biosynthetic pathway.