In this work the antiviral activity of 20 dehydroepiandrosterone (DHEA) analogs with different substituents at positions C-3, C-15, C-16 and C-17 were evaluated against vesicular stomatitis virus (VSV) in Vero cell cultures. The selectivity indexes (SI) obtained with DHEA and epiandrosterone (EA) were 50 and 72.6, respectively. The work showed that the compounds 21-norpregna-5,17(20)-dien-3beta,16alpha-diyl-diacetate, 17,17-ethylendioxyandrostan-5,15-dien-3beta-ol and 3beta-hydroxypregn-17(20)-en-16-one had higher SI values than ribavirin, which was used as a reference drug. The antiviral mode of action of DHEA was also investigated against VSV replication in Vero cells, and time of addition experiments showed that DHEA mainly affected a late event in the virus growth cycle. Analysis of RNA and protein synthesis indicated that DHEA adversely affected positive strand RNA synthesis and viral mature particle formation.