Androstene steroids are metabolites of dehydroepiandrosterone and exist as androstene-diols or -triols in alpha- and beta-epimeric forms based upon the placement of the hydroxyl groups relative to the plane of the Delta(5)cycloperhydrophenanthrene ring. 5-Androstene-3beta,17beta-diol (3beta,17beta-AED) functions to upregulate immunity and the addition of a third hydroxyl group at C-7 in the alpha- or beta-orientation (3beta,7alpha,17beta-AET and 3beta,7beta,17beta-AET, respectively) enhances the immunological activity of the molecule. In contrast, 5-androstene-3beta,17alpha-diol (3beta,17alpha-AED) possesses potent anti-tumor activity. We synthesized a new androstene by adding a third hydroxyl group at C-7 to make 5-androstene-3beta,7alpha,17alpha-triol (3beta,7alpha,17alpha-AET) and compared the anti-tumor activity of this steroid to the four existing androstenes. The results showed that this modification reduced the activity of 3beta,17alpha-AED. The ranking of the anti-tumor activities of these steroids and their IC50 on human glioblastoma and lymphoma cells was: 3beta,17alpha-AED ( approximately 10 microm) > 3beta,7alpha,17alpha-AET ( approximately 30 microm) " 3beta,7alpha,17beta-AET ( approximately 150 microm)> 3beta,7beta,17beta-AET (not achievable) >or= 3beta,17beta-AED (not achievable). 3beta,17alpha-AED and 3beta,7alpha,17alpha-AET induced autophagy in T98G glioblastoma cells and apoptosis in U937 lymphoma cells. These results indicate that the position of the hydroxyl group on C-17 dictates the anti-tumor activity of the androstenes and must be in the alpha-configuration, demonstrating a strict structure-activity relationship.