To elevate production of OF4949 byPeuicillium rugulosum OF4949 and to elucidate the pathway of its biosynthesis, mutants were selected on the basis of their resistance to growth inhibition by phenylalanine analogs. A mutant resistant tom-fluorophenylalanine, strain No. M414, had 3-fold the production of the parent. In a study of the biosynthesis of OF4949-I and II, several 14C-labeled compounds were examined as possible precursors of OF4949. L-[14C]Tyrosine and L-[14C]asparagine were incorporated efficiently. Most of the radioactivity of L-[14C]tyrosine was found in the 4-methylisodityrosine (B2) or isodityrosine (B1) moieties, and that of L-[14C]asparagine was in the β-hydroxyasparagine moiety.