Exogenous addition of D-serine to Actinomadura spinosa AA0851 resulted in directed production of pradimicin FS, a new D-serine analog of pradimicin S, together with pradimicin FB. Pradimicin FS was produced in higher yields by derivation of ferrous sulfate-resistant strains from strain AA0851. Pradimicin FB, a minor product, was identified as deglucosylpradimicin FL. Pradimicin FS was equivalent to pradimicin S in syncytium formation inhibition activity and in vitro and in vivo antifungal activities.