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The structures of atpenins A4, A5 and B, new antifungal antibiotics produced by Penicillium sp., have been deduced to be I, II and III, respectively, on the basis of spectroscopic and 1H and 13C NMR spectral data. The molecular structure of atpenin A4 with absolute configurations was finally confirmed to be 2'S,4'S,5'S-5,6-dimethoxy-4-hydroxy-5'-chloro-2',4'- dimethyl-1'-oxoheptyl-2-hydroxypyridine (I') by the single crystal X-ray crystallographic analysis. The absolute configurations of atpenins A5 and B were also expected to be 2'S,4'S,5'R-II (II') and 2'S,4'R-III (III'), respectively.