Further study of the methanol extract of the aerial parts of Physalis angulata (Solanaceae) resulted in the isolation of new withanolides, designated physagulins L, M and N, together with known withanolide, physagulin D and flavonol glycoside, quercetin 3-O-rhamnosyl-(1→6)-galactoside. The chemical structures of these new withanolides were elucidated by detailed spectroscopic analyses to be (20R,22R)-15α-acetoxy-5α,6β,14β,17β,27-pentahydroxy-1-oxo-witha-2, 24-dienolide, (20S,22S)-15α-acetoxy-5α,6β,14α,23β-tetrahydroxy-1-oxo-witha-2,16,24-trienolide and (20S,22R)-15α-acetoxy-5β,6β-epoxy-14α-hydoxy-3β-methoxy-1-oxo-witha-16,24-dienolide, respectively. All these compounds showed weak trypanocidal activity against trypomastigotes, an infectious form of Trypanosoma cruzi, the etiologic agent for Chagas' disease. Withanolides obtained in the previous paper showed considerable trypanocidal activity, suggesting the structure–activity relationships.