<jats:title>Abstract</jats:title> <jats:p>In this study, the antileukemic effects of three isomeric pregnadienedione steroids [i.e., cis-guggulsterone, trans-guggulsterone, and 16-dehydroprogesterone] were investigated in HL60 and U937 cells as well as in primary leukemic blasts in culture. Our results show that all three compounds inhibited the proliferation of HL60 and U937 cells, with IC50s ranging from 3.6 to 10.9 μmol/L after treatment for 6 days. These growth inhibitory effects correlated with externalization of phosphatidylserine and loss of mitochondrial membrane potential, suggesting that these isomeric steroids induce apoptosis in leukemia cells. z-VAD-fmk prevented phosphatidylserine externalization but not mitochondrial membrane potential loss, indicating that mitochondrial dysfunction occurred in the absence of caspase activation. Interestingly, although all three compounds increased the generation of reactive oxygen species and decreased phosphorylation of extracellular signal-regulated kinase, only cis-guggulsterone induced a rapid depletion of reduced glutathione levels and oxidation of the mitochondrial phospholipid cardiolipin. 16-Dehydroprogesterone and trans-guggulsterone induced differentiation of HL60 and NB4 cells as evidenced by increased surface expression of CD11b and/or CD14, and all three steroids rapidly induced mitochondrial dysfunction and phosphatidylserine externalization of CD34-positive blasts from primary leukemic samples. This study is the first to show that guggulsterones and 16-dehydroprogesterone exert antileukemic effects via the induction of apoptosis and differentiation and, more importantly, identifies the pregnadienedione structure as a potential chemotherapeutic scaffold. [Mol Cancer Ther 2005;4(12):1982–92]