HPLC bioautography of the directed biosynthesis of Zalerion arboricola led to the discovery of pneumocandin B0 (L-688, 786), a new antifungal and anti-Pneumocystis carinii lipopeptide. Isolation techniques were developed to separate this component from pneumocandin A0 (L-671, 329) in fermentations of a mutant of Zalerion arboricola. A number of related compounds were also isolated, which differ from pneurnocandins A0 and B0 in the hydroxylation patterns on the ornithine, homotyrosine, and proline.