Physalins D (1), B (2), F (3), 5α-ethoxy-6β-hydroxy-5,6-dihydrophysalin B (4) and E (5), all isolated from Physalis angulata L. extracts, were tested for cytotoxic activity using a panel of tumor cell lines and sea urchin eggs. In general, the most active compounds were 1 and 3, followed by 2 and 4. Compound 4a obtained by the hydrogenation of 4 was weaker than all the others in tumor cells and possessed no activity in sea urchin eggs. Compound 5 had no activity in tumor cells and exhibited activity in sea urchin eggs only in the third cleavage and blastulae. The present data suggest that the cytotoxic effect of physalins is probably determined by the combination of the conjugated cyclohexanone moiety and the presence of an oxygen located at both C- 5 and C-6.